Tricyclodecan-9-yl-xanthogenate (D609) mechanism of actions: a mini-review of literature.

Tricyclodecan-9-yl-xanthogenate (D609) is known for its antiviral and antitumor properties. D609 actions are widely attributed to inhibiting phosphatidylcholine (PC)-specific phospholipase C (PC-PLC). D609 also inhibits sphingomyelin synthase (SMS). PC-PLC and/or SMS inhibition will affect lipid second messengers 1,2-diacylglycerol (DAG) and/or ceramide. Evidence indicates either PC-PLC and/or SMS inhibition affected the cell cycle and arrested proliferation, and stimulated differentiation in various in vitro and in vivo studies. Xanthogenate compounds are also potent antioxidants and D609 reduced Aß-induced toxicity, attributed to its antioxidant properties. Zn²âº is necessary for PC-PLC enzymatic activity; inhibition by D609 might be attributed to its Zn²âº chelation. D609 has also been proposed to inhibit acidic sphingomyelinase or down-regulate hypoxia inducible factor-1α; however these are down-stream events related to PC-PLC inhibition. Characterization of the mammalian PC-PLC is limited to inhibition of enzymatic activity (frequently measured using Amplex red assay with bacterial PC-PLC as a standard). The mammalian PC-PLC has not been cloned; sequenced and structural information is unavailable. D609 showed promise in cancer studies, reduced atherosclerotic plaques (inhibition of PC-PLC) and cerebral infarction after stroke (PC-PLC or SMS). D609 actions as an antagonist to pro-inflammatory cytokines have been attributed to PC-PLC. The purpose of this review is to comprehensively evaluate the literature and summarize the findings and relevance to cell cycle and CNS pathologies.

Altered lipid metabolism in brain injury and disorders.

Deregulated lipid metabolism may be of particular importance for CNS injuries and disorders, as this organ has the highest lipid concentration next to adipose tissue. Atherosclerosis (a risk factor for ischemic stroke) results from accumulation of LDL-derived lipids in the arterial wall. Pro-inflammatory cytokines (TNF-alpha and IL-1), secretory phospholipase A2 IIA and lipoprotein-PLA2 are implicated in vascular inflammation. These inflammatory responses promote atherosclerotic plaques, formation and release of the blood clot that can induce ischemic stroke. TNF-alpha and IL-1 alter lipid metabolism and stimulate production of eicosanoids, ceramide, and reactive oxygen species that potentiate CNS injuries and certain neurological disorders. Cholesterol is an important regulator of lipid organization and the precursor for neurosteroid biosynthesis. Low levels of neurosteroids were related to poor outcome in many brain pathologies. Apolipoprotein E is the principal cholesterol carrier protein in the brain, and the gene encoding the variant Apolipoprotein E4 is a significant risk factor for Alzheimer's disease. Parkinson's disease is to some degree caused by lipid peroxidation due to phospholipases activation. Niemann-Pick diseases A and B are due to acidic sphingomyelinase deficiency, resulting in sphingomyelin accumulation, while Niemann-Pick disease C is due to mutations in either the NPC1 or NPC2 genes, resulting in defective cholesterol transport and cholesterol accumulation. Multiple sclerosis is an autoimmune inflammatory demyelinating condition of the CNS. Inhibiting phospholipase A2 attenuated the onset and progression of experimental autoimmune encephalomyelitis. The endocannabinoid system is hypoactive in Huntington's disease. Ethyl-eicosapetaenoate showed promise in clinical trials. Amyotrophic lateral sclerosis causes loss of motorneurons. Cyclooxygenase-2 inhibition reduced spinal neurodegeneration in amyotrophic lateral sclerosis transgenic mice. Eicosapentaenoic acid supplementation provided improvement in schizophrenia patients, while the combination of (eicosapentaenoic acid + docosahexaenoic acid) provided benefit in bipolar disorders. The ketogenic diet where >90% of calories are derived from fat is an effective treatment for epilepsy. Understanding cytokine-induced changes in lipid metabolism will promote novel concepts and steer towards bench-to-bedside transition for therapies.

Effect of tricyclodecan-9-yl potassium xanthate (D609) on phospholipid metabolism and cell death during oxygen-glucose deprivation in PC12 cells.

Alterations in lipid metabolism play an integral role in neuronal death in cerebral ischemia. Here we used an in vitro model, oxygen-glucose deprivation (OGD) of rat pheochromocytoma (PC12) cells, and analyzed changes in phosphatidylcholine (PC) and sphingomyelin (SM) metabolism. OGD (4-8 h) of PC12 cells triggered a dramatic reduction in PC and SM levels, and a significant increase in ceramide. OGD also caused increases in phosphatidylcholine-phospholipase C (PC-PLC) and phospholipase D (PLD) activities and PLD2 protein expression, and reduction in cytidine triphosphate:phosphocholine cytidylyltransferase-alpha (CCTalpha, the rate-limiting enzyme in PC synthesis) protein expression and activity. Phospholipase A2 activity and expression were unaltered during OGD. Increased neutral sphingomyelinase activity during OGD could account for SM loss and increased ceramide. Surprisingly, treatment with PC-PLC inhibitor tricyclodecan-9-yl potassium xanthate (D609) aggravated cell death in PC12 cells during OGD. D609 was cytotoxic only during OGD; cell death could be prevented by inclusion of sera, glucose or oxygen. During OGD, D609 caused further loss of PC and SM, depletion of 1,2-diacylglycerol (DAG), increase in ceramide and free fatty acids (FFA), cytochrome c release from mitochondria, increases in intracellular Ca2+ ([Ca2+]i), poly-ADP ribose polymerase (PARP) cleavage and phosphatidylserine externalization, indicative of apoptotic cell death. Exogenous PC during OGD in PC12 cells with D609 attenuated PC, SM loss, restored DAG, attenuated ceramide levels, decreased cytochrome c release, PARP cleavage, annexin V binding, attenuated the increase in [Ca2+]i, FFA release, and significantly increased cell viability. Exogenous PC may have elicited these effects by restoring membrane PC levels. A tentative scheme depicting the mechanism of action of D609 (inhibiting PC-PLC, SM synthase, PC synthesis at the CDP-choline-1,2-diacylglycerol phosphocholine transferase (CPT) step and causing mitochondrial dysfunction) has been proposed based on our observations and literature.

Secretory phospholipase A2 IIA is up-regulated by TNF-alpha and IL-1alpha/beta after transient focal cerebral ischemia in rat.

Cerebral ischemia initiates an inflammatory response in the brain that is associated with the induction of a variety of cytokines, including tumor necrosis factor-alpha (TNF-alpha) and interleukin-1alpha/beta (IL-1alpha/beta) that contributes to stroke injury. Transient middle cerebral artery occlusion (tMCAO) in spontaneously hypertensive rat (SHR) resulted in significant increases in TNF-alpha and IL-1beta levels. We have previously demonstrated up-regulation of secretory phospholipase A2 IIA (sPLA2 IIA) mRNA and protein expression, increased PLA2 activity, and loss of phosphatidylcholine after 1-h tMCAO and 24-h reperfusion in SHR. Treatment with TNF-alpha antibody or IL-1 receptor antagonist significantly attenuated infarction volume, sPLA2 IIA protein expression, PLA2 activity and significantly restored phosphatidylcholine levels after tMCAO. This suggests that cytokine induction up-regulates sPLA2 IIA protein expression, resulting in altered lipid metabolism that contributes to stroke injury.

Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia.

Ischemic stroke is caused by obstruction of blood flow to the brain, resulting in energy failure that initiates a complex series of metabolic events, ultimately causing neuronal death. One such critical metabolic event is the activation of phospholipase A2 (PLA2), resulting in hydrolysis of membrane phospholipids and release of free fatty acids including arachidonic acid, a metabolic precursor for important cell-signaling eicosanoids. PLA2 enzymes have been classified as calcium-dependent cytosolic (cPLA2) and secretory (sPLA2) and calcium-independent (iPLA2) forms. Cardiolipin hydrolysis by mitochondrial sPLA2 disrupts the mitochondrial respiratory chain and increases production of reactive oxygen species (ROS). Oxidative metabolism of arachidonic acid also generates ROS. These two processes contribute to formation of lipid peroxides, which degrade to reactive aldehyde products (malondialdehyde, 4-hydroxynonenal, and acrolein) that covalently bind to proteins/nucleic acids, altering their function and causing cellular damage. Activation of PLA2 in cerebral ischemia has been shown while other studies have separately demonstrated increased lipid peroxidation. To the best of our knowledge no study has directly shown the role of PLA2 in lipid peroxidation in cerebral ischemia. To date, there are very limited data on PLA2 protein by Western blotting after cerebral ischemia, though some immunohistochemical studies (for cPLA2 and sPLA2) have been reported. Dissecting the contribution of PLA2 to lipid peroxidation in cerebral ischemia is challenging due to multiple forms of PLA2, cardiolipin hydrolysis, diverse sources of ROS arising from arachidonic acid metabolism, catecholamine autoxidation, xanthine oxidase activity, mitochondrial dysfunction, activated neutrophils coupled with NADPH oxidase activity, and lack of specific inhibitors. Although increased activity and expression of various PLA2 isoforms have been demonstrated in stroke, more studies are needed to clarify the cellular origin and localization of these isoforms in the brain, their responses in cerebral ischemic injury, and their role in oxidative stress.

Schedule-dependent variations in the response of murine P388 leukemia to cyclophosphamide in combination with interferons-alpha/beta.

Positive therapeutic effects of interferons (IFNs) in combination with other therapies will depend on defining modalities, doses, and timing of treatment in the setting of varied tumor burdens. When 10(4) P388 leukemia cells were inoculated i.p. on day 0 in BALB/c x DBA/2 F1 mice, all mice died within 18 days if left untreated. Murine IFN-alpha/beta (5 x 10(5) units) injected daily i.p. on days 5-9 resulted in 20% increase in life span (ILS) (P less than 0.0001). Cyclophosphamide (CY) (100, 33, or 15 mg/kg) was injected i.p. once 2 days before start (day 3), simultaneously with start (day 5), or 2 days after cessation of IFN treatment (day 11). When 100 mg/kg CY alone were injected on day 3 or 5, all mice survived more than 90 days and were considered cured. When IFN was given after this curative dose of CY, more tumor deaths occurred; up to 100% of the mice died when 100 mg/kg CY on day 3 were combined with IFN on days 5-9. Increased mortality with the combination was not due to added toxicity of CY and IFN since the mice developed abdominal tumors and ascites. Mice not inoculated with tumor cells and treated similarly suffered only a transient weight loss, had only moderate white count depression, and did not die. When IFN was injected before CY on days 1-5 (instead of days 5-9), IFN did not alter the effectiveness of CY (100 mg/kg on day 5). In contrast to these results, when CY (100 mg/kg) was administered on day 11, after IFN (days 5-9), an augmented survival occurred with 119% ILS and 40% cures (CY alone on day 11 resulted in 69% ILS but no cures). In addition, when CY at a lower dose of 15 mg/kg was injected in combination with IFN, survival was consistently augmented by IFN; e.g., CY alone on day 3 caused 40% ILS and with IFN (days 5-9) 60% ILS (P less than 0.0001). Qualitatively similar findings were obtained when P388 leukemia cells were inoculated s.c. and the drugs delivered i.p. Inhibition by IFN of antitumor effects of a second alkylating agent, 1,3-bis(2-chloroethyl)-1-nitrosourea, was also identified. Thus, IFN-alpha/beta potentiated suboptimal CY effects for P388 leukemia, had neutral effects when injected before CY treatment, and inhibited antitumor activity of curative CY or nitrosourea schedules.

Quercetin, a rat intestinal and bladder carcinogen present in bracken fern (Pteridium aquilinum).

Albino noninbred weanling male and female rats were fed a basic grain diet (Group 1) or a basic diet supplemented with 33% bracken fern [BF (Group 2)] or 0.1% quercetin [purity, > 99% (Group 3)] for 58 weeks. The quantities of quercetin and kaempferol (a close structural analog) in BF as glycosides were determined to be 0.57 and 1.1 g, respectively, per kg of dried BF. Estimated mean total cumulative doses (mmol) per rat were: Group 1, quercetin, males and females < 0.03; kaempferol, males and females < 0.03; Group 2, quercetin, males 5.8, females 5.2; kaempferol, males 11.9, females 10.8; and Group 3, quercetin, males 27.8, females 25.3; kaempferol, males and females < 0.03. Growth of rats fed BF or quercetin was comparable but significantly (p < 0.01) slower after 24 weeks than that of Group 1. Mean survivals (weeks) of rats of all groups were: Group 1, 58 +/- 7 (S.D.); Group 2, 51 +/- 13; and Group 3, 56 +/- 8. They were not significantly different, although rats fed BF tended to die earlier secondary to intestinal tumor-induced intussusception and obstruction. The following incidences of intestinal or bladder neoplasms in male or female rats, respectively, were observed: Group 1, intestinal and bladder, males, 0 of 9, females, 0 of 10; Group 2, intestinal, males, 7 of 8, females, 10 of 11; bladder, males, 6 of 8, females 8 of 11; Group 3, intestinal, males, 6 of 7, females, 14 of 18; bladder, males, 2 of 7, females, 3 of 18. The histopathology of neoplasms of the 2 target organs was identical for rats of Groups 2 and 3. Multiple ileal intestinal neoplasms of rats fed quercetin included: adenoma, 4; fibroadenoma, 7; and adenocarcinoma, 9 (with mesenteric metastases, 3). The 5 bladder tumors were papillary or sessile transitional cell carcinomas.

Inhibition of murine tumor growth by an interferon-inducing imidazoquinolinamine.

The low-molecular-weight imidazoquinolinamine derivative, 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine (imiquimod, previously described as R-837), induced alpha-interferon (IFN-alpha) in mice. IFN induction was identified at oral doses as low as 3 mg/kg. The 10% lethal dose for daily treatment with imiquimod was 200 mg/kg. Oral treatment with 30 mg/kg imiquimod once every three days significantly inhibited MC-26 colon carcinoma. Delay of treatment from day 1 to day 5, when tumors were easily palpable, did not reduce benefits. Ten daily treatments were slightly more effective than five. However, delivery of the same total dose of imiquimod either once every day for 20 days, once every 4 days, once every 7 days, or once every 10 days inhibited tumor growth to the same level. The antitumor effects of imiquimod were significantly abrogated by an antiserum to murine IFN-alpha, suggesting that the antitumor effect was to a substantial extent mediated by IFN induction. Imiquimod also significantly reduced the number of lung colonies in mice inoculated i.v. with MC-26 tumor cells. Combination of treatment with imiquimod and cyclophosphamide was significantly (P less than 0.01) better than treatment with either drug alone. Combination treatment with cyclophosphamide led to cures in some of the mice inoculated either s.c. or i.v. with MC-26 cells. Treatment with imiquimod also inhibited the growth of RIF-1 sarcoma and Lewis lung carcinoma but was ineffective for P388 leukemia. Imiquimod is an oral IFN-alpha inducer with antitumor effectiveness for transplantable murine tumors.

Effects of citicoline on phospholipid and glutathione levels in transient cerebral ischemia.

BACKGROUND AND PURPOSE: Cytidine-5'-diphosphocholine (citicoline or CDP-choline) is an essential intermediate in the biosynthesis of phosphatidylcholine, an important component of the neural cell membrane. Citicoline provided significant neuroprotection after transient forebrain ischemia in gerbils. This study was undertaken to examine changes and effects of citicoline on phospholipids and glutathione synthesis after transient cerebral ischemia and reperfusion. METHODS: Ten-minute transient forebrain ischemia was induced by bilateral carotid artery occlusion in male Mongolian gerbils with reperfusion up to 6 days. Citicoline (500 mg/kg IP in saline) was given to gerbils just after the end of ischemia, at 3-hour reperfusion, and daily thereafter until 1 day before euthanasia. Hippocampal lipids were extracted and analyzed by thin-layer and gas chromatography. Glutathione was measured by using an enzymatic recycling assay. Glutathione reductase activity was determined by measuring NADPH oxidation. RESULTS: Significant decreases in phospholipids occurred at 1-day reperfusion. Citicoline significantly restored the phosphatidylcholine, sphingomyelin, and cardiolipin levels but did not affect phosphatidylinositol and phosphatidylserine at 1 day. The phospholipids returned to sham levels over days 2 to 6 and were unaffected by citicoline. Ceramide levels significantly increased by 3 and 6 days of reperfusion and were unaltered by citicoline. Ischemia resulted in significant decreases in glutathione and glutathione reductase activity over 3 days of reperfusion. Citicoline significantly increased total glutathione and glutathione reductase activity and decreased the glutathione oxidation ratio, an indicator of glutathione redox status. CONCLUSIONS: Our data indicated that the effects of citicoline on phospholipids occurred primarily during the first day of reperfusion, with effects on glutathione being important over the 3-day reperfusion period.

Loss of cardiolipin and mitochondria during programmed neuronal death: evidence of a role for lipid peroxidation and autophagy.

Cardiolipin, a lipid of the mitochondrial inner membrane, is lost from many types of cells during apoptotic death. Here we show that the cardiolipin content of nerve growth factor (NGF)-deprived rat sympathetic neurons undergoing apoptotic death in cell culture decreased before extensive loss of mitochondria from the cells. By 18-24 h after NGF deprivation, many neurons did not stain with the cardiolipin-specific dye, Nonyl Acridine Orange, suggesting complete loss of cardiolipin. Gas chromatography confirmed the decline of cardiolipin content in NGF-deprived neurons. Electron microscopy and immunoblots for the mitochondrial-specific protein, heat shock protein 60 (HSP60), revealed that there was only a slight decrease in mitochondrial mass at this time. Cardiolipin loss after NGF deprivation was concurrent with increased production of mitochondrial-derived reactive oxygen species [Kirkland, R.A., Franklin, J.L., 2001. J. Neurosci. 21, 1949-1963] and increased lipid peroxidation. Compounds having antioxidant effects blocked peroxidation, loss of cardiolipin, and the decrease of mitochondrial mass in NGF-deprived neurons. These compounds also blocked an increase in the number of lysosomes and autophagosomes in NGF-deprived cells. The findings reported here show that the important mitochondrial inner membrane lipid, cardiolipin, is lost from mitochondria during neuronal apoptosis and that this loss occurs before significant loss of mitochondria from cells. They suggest that the loss of cardiolipin is mediated by free radical oxygen.

Does CDP-choline modulate phospholipase activities after transient forebrain ischemia?

Ten min forebrain ischemia/1-day reperfusion resulted in significant decreases in total phosphatidylcholine (PtdCho), phosphatidylinositol (PtdIns), and cardiolipin in gerbil hippocampus. CDP-choline restored cardiolipin levels, arachidonic acid content of PtdCho, partially but significantly restored total PtdCho, and had no effect on PtdIns. These data suggest that CDP-choline prevented the activation of phospholipase A(2) (rather than inhibiting phospholipase A(2) activity) but did not affect activities of PtdCho-phospholipases C and/or D, or phosphoinositide-phospholipase C. CDP-choline also provided significant protection for hippocampal CA(1) neurons.

Fluorometric assay of nitrite and nitrate in brain tissue after traumatic brain injury and cerebral ischemia.

Nitric oxide synthase (NOS) is distributed within the brain, and nitric oxide (NO) is felt to be involved in the pathophysiology of deterioration after head injury and cerebral ischemia. This study determined the levels of the stable end products of NOS (NOx=nitrite+nitrate) after traumatic brain injury (TBI) and transient cerebral ischemia. A fluorometric assay using nitrate reductase and the NADPH regenerating system was used to quantitate NOx in ultrafiltered (10-kDa cutoff) cortical and hippocampal extracts after reduction of nitrate. In TBI rats, both the plasma and tissue showed a sharp increase in NOx levels 5 min after injury. Plasma NOx returned to control levels by 2 h after injury. Ipsilateral-cortex NOx levels returned to control levels approximately 6 h after injury and remained constant from 6-24 h. Contralateral-cortex returned near to control levels after 1 h. Hippocampus also followed a similar trend. In gerbils, there was a significant elevation in tissue NOx levels immediately after 10 min transient cerebral ischemia, which gradually returned to control levels over 24 h reperfusion. This striking burst of NO synthesis immediately after injury is clearly evident whether the injury is head trauma or ischemia, or whether the measurements were performed on tissue or plasma. It is unknown whether endothelial NOS, neuronal NOS, or both caused the elevation of the NO end products seen after the CNS insults.

Comparison of acyltransferase-mediated mutagenicity and nucleic acid binding of N-acetoxy-4-acetylaminobiphenyl by hepatic and bladder microsomes from rats and dogs.

Acyltransferase-mediated mutagenic and metabolic activation of N-acetoxy-4-acetylaminobiphenyl (N-OAc-AABP) by hepatic tissues of rats and dogs were compared. N-OAc-AABP was mutagenic in Salmonella typhimurium TA98 even in the absence of exogenous enzyme(s). However, supplementation with hepatic microsomes from dogs showed a dose-dependent increase in mutagenicity of N-OAc-AABP, whereas under the same conditions, rat microsomes were inactive. Incubation of liver microsomes with RNA showed that 46.4 and 11.2 nmole of [3H]N-OAc-AABP were bound/mg RNA/mg protein with dogs and rats, respectively. The hepatic microsome-mediated binding and mutagenicities of N-OAc-AABP were blocked by paraoxon, suggesting the involvement of deacetylase(s) in the activation process. Analyses of the in vitro incubates of N-OAc-AABP with rat and dog liver microsomes revealed the O-deacetylation product N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP) as the major metabolite. The ratios of O-deacetylation of N-O[14C]Ac-AABP versus N-deacetylation of N-OAc-[14C]AABP for hepatic microsomes from dogs and rats were 2.9 and 7.2, respectively. The O- and N-deacetylases are also distributed in bladder tissues and their activities in comparison to the hepatic tissues were lower and amounted to 14.2 and 5.0 nmoles (O/N-deacetylation ratio 2.8) for dogs and 14.8 and 1.7 nmoles per mg protein per min (O/N-ratio of 8.7) for rats. The microsomes from bladder tissues also catalyzed the binding of [3H]N-OAc-AABP to RNA and enhanced its mutagenic response in TA98, both of which were blocked by paraoxon. The occurrence of deacetylase(s) in the target tissues of the bladder carcinogen 4-acetylaminobiphenyl (AABP) suggests that metabolic activation of some of the proximate metabolites could occur within these target organs. Furthermore, since the O-deacetylation product N-OH-AABP is relatively innocuous compared to the N-deacetylation product N-acetoxy-4-aminobiphenyl, these results imply that the refractiveness of rats for 4-aminobiphenyl or AABP-induced bladder carcinogenesis might in part be associated with the higher ratios of microsomal O/N-deacetylase activities. Thus susceptibility to arylamine or arylacetamide-induced liver and bladder carcinogenesis might be influenced by the microsomal deacetylases.

Metabolic reduction of novel 3,4-dichloro-5-nitrofurans in Salmonella typhimurium.

To gain insight on biochemical mechanisms of mutagenesis and carcinogenesis by the experimental carcinogens, 5-nitrofurans, a new series of 3,4-dichloro-5-nitrofurans, comprised of 3,4-dichloro-5-nitro-2-acetylfuran (I), 3,4-dichloro-5-nitro-2-bromoacetylfuran (II), methyl 3,4-dichloro-5-nitro-2-furoate (III), were synthesized and tested for their activation to mutagenic forms in the standard plate assay using Salmonella typhimurium TA98, TA100, and TA100NR, a derivative of TA100 deficient in nitroreductase activity. The mutagenic responses in TA98 were 2- to 6-fold lower compared to TA100. Furthermore, I and II were less active in TA100NR, while compound III was about four times more mutagenic in TA100NR compared to the parent strain TA100. Incubation of III with NADPH and bacterial lysates showed that the extent of reduction was greater in TA100 compared to TA100NR. High-pressure liquid chromatography analysis of the ethyl acetate extract obtained from incubation of III with lysates of TA100 revealed the formation of four metabolites with retention times of about 4.0, 5.7, 10.0, and 14.3 minutes. The spectroscopic and chromatographic properties of the components with retention times of 10.0 and 14.3 minutes were identical to two derivatives obtained by chemical reduction of III, and thus represent nitroreduction products. These derivatives have been identified as cis- and trans-oxime isomers of methyl 3,4-dichloro-2-furoate, based on spectroscopic analyses. These oximes were not mutagenic for TA100. Furthermore, III was more mutagenic under anaerobic conditions, suggesting that secondary superoxide or nitroanion free radicals generated from nitroreduction are not responsible for the mutagenicity of III. In addition, the higher mutagenic response in TA100NR, and the lack of mutagenic activities of the amino and the oxime analogs of III suggest that the mutagenic activation of III might be due to the nitroso intermediate or involve mechanisms other than nitroreduction.

Mutagenicity of N- and O-acetyl derivatives of methyl 3,4-diphenyl-5-hydroxylamino-2-furoate and N-hydroxy-4-aminobiphenyl in Salmonella typhimurium.

Methyl 3,4-diphenyl-5-hydroxylamino-2-furoate (N-OH-MDPF) (I), methyl 3,4-diphenyl-5-acetoxyamino-2-furoate (N-OAc-MDPF) (II), methyl 3,4-diphenyl-N-hydroxy-5-acetylamino-2-furoate (N-OH-MDPAF) (III), and methyl 3,4-diphenyl-N-acetoxy-5-acetylamino-2-furoate (N-OAc-MDPAF) (IV) were synthesized and tested for mutagenic activity for Salmonella typhimurium TA98 and TA100. The hydroxylamine (I) and acetyl derivatives (II-IV) did not show mutagenic activity in TA98 or TA100. In contrast, the parent nitro compound, methyl 3,4-diphenyl-5-nitro-2-furoate (MDPNF) (V) was found to be equally active in TA98 and TA98-DNP, and more active in TA100 and TA104. The mutagenic activity in TA100 and TA104 decreased significantly under anaerobic conditions. Additionally, MDPNF was previously shown to be less mutagenic in the nitroreductase-deficient derivatives TA100NR and TA98NR, suggesting a requirement for nitro reduction. Incubation of V with NADPH and bacterial lysates of TA98 or TA98NR yielded a metabolite which was identified as I based on chromatographic and mass spectral characteristics. The rate of reduction by the lysate of TA98NR was about one-third that of TA98, showing a correlation between mutagenicity and nitroreductase activity. The lysates of TA98 did not reduce N-OH-MDPF further to the amine. In contrast to the lack of mutagenic activity of I-IV, N-hydroxy-4-aminobiphenyl (N-OH-ABP) and its acetyl derivatives were active in TA98, but less so in TA98-DNP. These data suggest that mechanisms involving O-acetylation of N-hydroxylamine to the acetoxyamine or acyl transfer reactions are not involved in the generation of mutagen from MDPNF. Furthermore, the differential mutagenic response of V in TA98 and TA98NR, its reduction to I, and the lack of activity of I suggest that the intermediates of reduction between the nitro and hydroxylamine, such as nitro or nitroso free radical anions, may be involved in mutagenesis. The decreased response of V under anaerobic conditions and increased response in TA104 suggest that secondary oxygen radicals generated from reduction intermediates may be responsible for the mutagenicity of MDPNF.

Neuroprotection by group I metabotropic glutamate receptor antagonists in forebrain ischemia of gerbil.

Stimulation of group I metabotropic glutamate receptors (mGluR 1 and 5) activates G-protein coupled-phospholipase C (PLC) to release 1,2-diacylglycerol (DAG) and arachidonic acid (ArAc). To elucidate the role of group I mGluR, we tested the effects of (S)-alpha-methyl-4-carboxy-phenylglycine (MCPG, mGluR 1 and 5 antagonist), 1-aminoindan-1,5-dicarboxylic acid (AIDA, mGluR 1a specific antagonist) and 2-methyl-6-(phenylethynyl) pyridine (MPEP, mGluR 5 antagonist) on ArAc release and neuronal survival after transient forebrain ischemia in gerbils. Ischemia resulted in (a) significant release of ArAc at 1-day reperfusion and (b) significant neuronal death in the hippocampal CA1 subfield after 6-day reperfusion. MCPG and MPEP decreased ArAc release and also significantly increased neuronal survival. AIDA was less effective in decreasing ArAc release and had no effect on neuronal death. These results suggest that activation of mGluR 5 may be an important pathway in ArAc release and neuronal death after transient ischemia.

Simultaneous assay of ornithine decarboxylase and polyamines after central nervous system injury in gerbil and rat.

Ornithine decarboxylase (ODC) is considered the rate-limiting enzyme in polyamine biosynthesis. An increase in putrescine (a natural polyamine) synthesis after central nervous system (CNS) injury appears to be involved in blood-brain barrier dysfunction, development of vasogenic edema and neuronal death. An improved method is described to determine the ODC activity as well as polyamine levels from the same brain tissue. The polyamine results showed no significant differences from data obtained with the conventional assay. The advantages of this method are to: (1) minimize the number of animals needed for the study, and (2) eliminate any internal inconsistencies resulting from use of two independent groups of animals for ODC and polyamine measurements. Using this method, ODC activities and polyamine levels were measured in cortices and hippocampi from global transient ischemia of gerbils and traumatic brain injury (TBI) of rats.

Xanthine oxidase-mediated mutagenicity of the bladder carcinogen 4-nitrobiphenyl.

Xanthine oxidase catalyzed mutagenicity of 4-nitrobiphenyl (NBP), a dog-bladder carcinogen, was tested in Ames assay using Salmonella typhimurium TA98 strains. NBP was active as a mutagen in the parent strain TA98 which is proficient in nitroreductase, while it was inactive in the strain TA98NR which is deficient in nitroreductase. However, preincubation of NBP at 37 degrees C with NADH and commercial preparations of xanthine oxidase for 30 min resulted in a dose-dependent increase in the mutagenic activity in TA98NR. Allopurinol blocked the xanthine oxidase catalyzed mutagenicity of NBP in TA98NR and the extent of inhibition was dependent upon the concentration of the inhibitor. Rat-liver and dog-bladder cytosol preparations also enhanced the mutagenic activity of NBP in TA98NR in a dose-dependent manner. In addition, the cytosol-mediated activity was also inhibited by allopurinol, implying that the cytosolic enzyme activity might be due to xanthine oxidase. In vitro enzymatic reduction of NBP using bacterial cell lysates of TA98 and TA98NR revealed the major product of reduction to be 4-aminobiphenyl. The transient intermediates of reduction were not detected during the in vitro incubation. The reduction intermediate N-hydroxylaminobiphenyl showed direct and equal mutagenic activity in both TA98 and TA98NR, in contrast to NBP. These results suggest that N-hydroxylaminobiphenyl is generated during the preincubation of NBP with xanthine oxidase or cytosolic preparations and the former might account for the mutagenicity of NBP. Furthermore, the occurrence of such enzyme(s) in the target tissue for NBP carcinogenesis, support the hypothesis that metabolic activation of the bladder carcinogen NBP could occur within the target organ by virtue of its intrinsic metabolic potential.

Factors affecting the mutagenic activity of quercetin for Salmonella typhimurium TA98: metal ions, antioxidants and pH.

The mutagenic activity of quercetin for Salmonella typhimurium TA98 was inhibited by addition of metal salts. MnCl2 was a potent inhibitor, followed by CuCl2, FeSO4, and FeCl3, the probable mechanism being facilitated catalytic oxidation of quercetin. With quercetin incorporated at a level of 100 nmoles/plate, approximate doses (nmoles/plate) to give 50% inhibition of mutagenic activity were: MnCl2 less than 10 (-S9), 18 (+S9); CuCl2 65 (-S9), greater than 100 (+S9); FeSO4 190 (-S9), greater than 300 (+S9); or FeCl3 275 (-S9), greater than 300 (+S9). Ascorbate, superoxide dismutase, and, to a lesser extent, NADH and NADPH, all enhanced the mutagenic activity of quercetin in the absence of the mammalian-microsome (S9) system, but had no significant effect in the presence of the S9 mix. The maximum enhancement of activity by ascorbate or superoxide dismutase was approximately 87% of the increase achieved by addition of the S9 mix. Tyrosinase (catechol oxidase) substantially reduced the mutagenic activity of quercetin in the absence of the S9 mix. At lower levels of tyrosinase, activity was restored by incorporation of the S9 mix. It is proposed that the S9 mix enhances the mutagenic activity of quercetin by scavenging superoxide radicals, thus inhibiting the autoxidation of quercetin, and possibly by reducing quinone oxidation products of quercetin. The mutagenic activity of quercetin increased substantially when the pH of the media was decreased. This may be due in part to a decrease in ionization of quercetin at lower pH, thereby increasing its absorption by the tester strain, to a decrease in the rate of autoxidation of quercetin at lower pH, or to a combination of these.

Phospholipase A(2), reactive oxygen species, and lipid peroxidation in CNS pathologies.

The importance of lipids in cell signaling and tissue physiology is demonstrated by the many CNS pathologies involving deregulated lipid metabolism. One such critical metabolic event is the activation of phospholipase A(2) (PLA(2)), which results in the hydrolysis of membrane phospholipids and the release of free fatty acids, including arachidonic acid, a precursor for essential cell-signaling eicosanoids. Reactive oxygen species (ROS, a product of arachidonic acid metabolism) react with cellular lipids to generate lipid peroxides, which are degraded to reactive aldehydes (oxidized phospholipid, 4-hydroxynonenal, and acrolein) that bind covalently to proteins, thereby altering their function and inducing cellular damage. Dissecting the contribution of PLA(2) to lipid peroxidation in CNS injury and disorders is a challenging proposition due to the multiple forms of PLA(2), the diverse sources of ROS, and the lack of specific PLA(2) inhibitors. In this review, we summarize the role of PLA(2) in CNS pathologies, including stroke, spinal cord injury, Alzheimer's, Parkinson's, Multiple sclerosis-Experimental autoimmune encephalomyelitis and Wallerian degeneration.