Can dicoumarol be used as a gonad-safe anticancer agent: an in vitro and in vivo experimental study.

STUDY HYPOTHESIS: Dicoumarol (DC) has potential for use as a gonad-safe anticancer agent. STUDY FINDING: DC altered cell proliferation, decreased viability and increased apoptosis in Vero and MCF-7 cell lines but did not show any toxic effect on mouse ovarian tissues and developing oocytes in vitro and in vivo. WHAT IS KNOWN ALREADY: DC suppresses cell proliferation and increases apoptosis in various cancer cells such as breast, urogenital and melanoma. DC has also been reported to alter the anticancer effects of several chemotherapeutics, including cisplatin, gemcitabine and doxorubicin in prostate, liver and uroepithelial cancer cells, respectively. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Vero (African green monkey kidney epithelial cells) and MCF-7 (human cancerous breast epithelial cells) cell lines and mouse granulosa cells isolated from 21-day-old female BALB/c mice (n = 21) were used to assess the effects of DC (10, 50, 100 and 200 µm) for 24 and 48 h on cell proliferation, viability and apoptotic cell death. In vivo experiments were performed with a single i.p. injection of 32 mg/kg DC in 21-day-old female BALB/c mice (n = 12). Following 48 h, animals were sacrificed by cervical dislocation and histological sections of isolated ovaries were evaluated for apoptosis. Viability assays were based on the trypan blue dye exclusion method and an automated cell counter device was used. Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and Annexin-V immunofluorescence were assessed by 3D confocal microscopy to address apoptotic cell death. We also assessed whether DC inhibits cell proliferation and viability through NQO1 [NAD(P)H Quinone Oxidoreductase 1], an intracellular inhibitor of reactive oxygen species (ROS). The meiotic spindle and chromosomes were studied in mouse oocytes by α-ß-tubulin and 7-aminoactinomycine D (7-AAD) immunostaining in vitro and in vivo. MAIN RESULTS AND THE ROLE OF CHANCE: DC does not block oocyte maturation and no significant alteration was noted in meiotic spindle or chromosome morphology in metaphase-II (M-II) stage oocytes following DC treatment in vitro or in vivo. In contrast, exposure to DC for 24 h suppressed cell proliferation (P = 0.026 at 200 µm), decreased viability (P = 0.002 at 200 µm) and increased apoptosis (P = 0.048 at 100 µm) in Vero and MCF-7 cell lines, compared with controls. These changes were not related to intracellular NQO1 levels. Mouse granulosa cells were unaffected by 50 or 100 µm DC treatment for 24 and 48 h in vitro. DC treatment in vivo did not alter the number of primordial follicles or the ratio of apoptosis in primordial, primary and secondary follicles, as well as in antral follicles, compared with the controls. LIMITATIONS, REASONS FOR CAUTION: DC was tested for ovarian toxicity only in isolated mouse oocytes/ovaries and healthy BALB/c mice. No cancer formation was used as an in vivo test model. The possibility that DC may potentiate ovarian toxicity when combined with traditional chemotherapeutic agents, such as mitomycin-C, cisplatin, gemcitabine and doxorubicin, must be taken into account, as DC is known to alter their effects in some cancer cells. WIDER IMPLICATIONS OF THE FINDINGS: The present study evaluated, for the first time, the effect of DC on ovarian tissue. The results suggested that DC is not toxic to ovarian tissues and developing oocytes; therefore, DC should be assessed further as a potential anticancer agent when female fertility preservation is a concern. LARGE SCALE DATA: N/A. STUDY FUNDING AND COMPETING INTERESTS: This work includes data from dissertation thesis entitled 'Effects of dicoumarol on mitotic and meiotic cells as an anticancer agent' by DA, 2014 and was partly supported by The National Scientific and Technological Research Council of Turkey (SBAG-109S415) to AC, OC and SO. The authors confirm that this article content presents no conflicts of interest.

Synthesis, antibacterial activities, and theoretical studies of dicoumarols.

Four dicoumarols (DC, 2-PyDC, 3-PyDC and 4-PyDC) were synthesized and characterized via IR, (1)H NMR, HRMS, and single crystal X-ray crystallography. Two classical intramolecular O-H···O hydrogen bonds (HBs) stabilized their structures. The total HB energies in DC, 2-PyDC, 3-PyDC and 4-PyDC were calculated with the density functional theory (DFT) [B3LYP/6-31G*] method. The in vitro antibacterial activity of DC, 2-PyDC, 3-PyDC and 4-PyDC against Staphylococcus aureus (S. aureus ATCC 29213), methicillin-resistant S. aureus (MRSA XJ 75302), vancomycin-intermediate S. aureus (Mu50 ATCC 700699), and USA 300 (Los Angeles County clone, LAC) was evaluated by observing the minimum inhibitory concentration and time-kill curves. The results showed that among all the compounds, 2-PyDC exhibited the most potent antibacterial activity.

Prodrug strategy to achieve lyophilizable, high drug loading micelle formulations through diester derivatives of ß-Lapachone.

ß-Lap prodrug micelle strategy improves the formulation properties of ß-lap therapeutics. The resulting micelles yield apparent high ß-lap solubility (>7 mg mL(-1) ), physical stability, and ability to reconstitute after lyophilization. In the presence of esterase, ß-lap prodrugs are efficiently converted into parent drug (i.e., ß-lap), resulting in NQO1-dependent lethality of NSCLC cells.

Protective effects of nicotine against aminochrome-induced toxicity in substantia nigra derived cells: implications for Parkinson's disease.

Parkinson's disease is a debilitating progressive neurodegenerative disorder that results from the loss of or damage to dopaminergic cells containing neuromelanin in the substantia nigra (SN). The underlying neurodegenerative mechanism(s), however, remain elusive. Aminochrome, the precursor of neuromelanin is an endogenous substance capable of inducing selective neurotoxicity to dopaminergic neurons in SN. Nicotine, on the other hand, may offer protective effects against dopaminergic cell damage induced by various neurotoxins including MPTP and salsolinol. In this study, we sought to determine whether nicotine may also protect against aminochrome-induced toxicity in SN derived RCSN-3 cells. Exposure of RCSN-3 cells to a combination of aminochrome (50 µM) and dicoumarol (50 µM) for 48 h induced approximately 70 % cell death. Pretreatment with nicotine, dose-dependently blocked this toxicity. The effects of nicotine in turn were dose-dependently blocked by mecamylamine, a non-selective nicotinic receptor antagonist. These results suggest involvement of nicotinic receptors in protective effects of nicotine against aminochrome-induced toxicity and provide further evidence for possible therapeutic effects of nicotine or nicotinic agonists in Parkinson's disease.

Inhibitors of NQO1: identification of compounds more potent than dicoumarol without associated off-target effects.

The enzyme NAD(P)H quinone oxidoreductase (NQO1) can function both as a detoxifying enzyme as well as chaperone protein. The latter property has been extensively characterized by the use of dicoumarol which inhibits the chaperone properties of NQO1 in cells. However, the use of this compound is compromised by its multiple "off-target" effects. Coumarin-based compounds that are more potent than dicoumarol as inhibitors of NQO1 in cells have been identified (Nolan et al., Biochem Pharmacol 2010;80:977-81). The purpose of the work reported here is to evaluate the off-target effects of these compounds when compared to dicoumarol. A range of these substituted coumarins are identified that are significantly less toxic than dicoumarol in a panel of nine cell lines. Further a number of the compounds generate much less intracellular superoxide, and many of them also show a reduced ability to induce apoptosis when compared to dicoumarol. None of these effects correlate with the ability of the compounds to inhibit the enzymatic activity of NQO1 in cells. In conclusion, potent inhibitors of NQO1 have been identified that will be more pharmacologically useful than dicoumarol for probing the function of NQO1 in cells and tissues.

Copper neurotoxicity is dependent on dopamine-mediated copper uptake and one-electron reduction of aminochrome in a rat substantia nigra neuronal cell line.

The mechanism of copper (Cu) neurotoxicity was studied in the RCSN-3 neuronal dopaminergic cell line, derived from substantia nigra of an adult rat. The formation of a Cu-dopamine complex was accompanied by oxidation of dopamine to aminochrome. We found that the Cu-dopamine complex mediates the uptake of (64)CuSO(4) into the Raúl Caviedes substantia nigra-clone 3 (RCSN3) cells, and it is inhibited by the addition of excess dopamine (2 m M) (63%, p < 0.001) and nomifensine (2 microM) (77%, p < 0.001). Copper sulfate (1 m M) alone was not toxic to RCSN-3 cells, but was when combined with dopamine or with dicoumarol (95% toxicity; p < 0.001) which inhibits DPNH and TPNH (DT)-diaphorase. Electron spin resonance (ESR) spectrum of the 5,5-dimethylpyrroline-N-oxide (DMPO) spin trap adducts showed the presence of a C-centered radical when incubating cells with dopamine, CuSO(4) and dicoumarol. A decrease in the expression of CuZn-superoxide dismutase and glutathione peroxidase mRNA was observed when RCSN-3 cells were treated with CuSO(4), dopamine, or CuSO(4) and dopamine. However, the mRNA expression of glutathione peroxidase remained at control levels when the cells were treated with CuSO(4), dopamine and dicoumarol. The regulation of catalase was different since all the treatments with CuSO(4) increased the expression of catalase mRNA. Our results suggest that copper neurotoxicity is dependent on: (i) the formation of Cu-dopamine complexes with concomitant dopamine oxidation to aminochrome; (ii) dopamine-dependent Cu uptake; and (iii) one-electron reduction of aminochrome.

Hemorrhagic diathesis, mesenteric hematoma, and colic associated with ingestion of sweet vernal grass in a cow.

A 4-year-old Holstein cow from a farm where 2 cows had recently died suddenly was referred for evaluation of acute severe colic. Right flank laparotomy revealed a large mesenteric hematoma. Within 14 layer chromatographic analyses of the moldy hay and blood from the necropsied cow and the hospitalized cow were positive for dicumarol. A diagnosis of sweet vernal poisoning was confirmed on the basis of clinical and toxicologic findings. The cow was treated with supportive therapy, blood transfusions, and vitamin K1 and recovered without complications. Because sweet vernal grass is becoming common in certain areas and the use of round bales is commonplace, practitioners should be aware of the potential for this toxicosis.

Menadione causes increases in the level of glutathione and in the activity of gamma-glutamylcysteine synthetase in cultured Chinese hamster V79 cells.

Incubation of cultured Chinese hamster V79 cells with menadione (2-methyl-1,4-naphthoquinone), a generator of superoxide anion radicals, caused a rapid increase in the level of glutathione disulfide (GSSG) and a decrease in the level of glutathione (GSH), which followed a 1.5- to 2-fold increase in the level of GSH during post-treatment incubation. Menadione also caused a concentration- and time-dependent increase in the activity of gamma-glutamylcysteine synthetase (gamma-GCS), a rate-limiting enzyme in the synthesis of GSH. These results suggested that the increase in level of GSH after treatment with menadione was due to the increase in the activity of gamma-GCS. Dicoumarol, an inhibitor of DT-diaphorase, did not influence the increase in the activity of gamma-GCS caused by menadione but it did enhance the cytotoxicity and the increase in the level GSSG caused by menadione. This result suggested that neither the DT-diaphorase-mediated metabolism of menadione nor the increase in level of GSSG caused by menadione was associated with the increase in the activity of gamma-GCS. Chelators of divalent iron and copper (I), and cycloheximide did not influence the increase in the activity of gamma-GCS caused by menadione. Thus, it appeared that reactive oxygen radicals, generated from hydrogen peroxide by an iron- or copper-catalyzed Fenton reaction, were not responsible for the increase in the activity of gamma-GCS and that the increase was not an inducible phenomenon.

Menadione toxicity in two mouse liver established cell lines having striking genetic differences in quinone reductase activity and glutathione concentrations.

Established cell lines derived from newborn livers of c14CoS/c14CoS and cch/cch mice were examined for differences in menadione toxicity. The 14CoS/14CoS cells exhibit 10-fold higher NAD(P)H:menadione oxidoreductase (NMO1) activity and 3-fold greater concentrations of reduced glutathione (GSH) than the ch/ch cells. In 14CoS/14CoS cells there are also 50% to 3-fold increases in glutathione transferase (GSTA1), UDP glucuronosyltransferase, and the copper, zinc-dependent superoxide dismutase activities. Catalase activity, on the other hand, is six times lower in the 14CoS/14CoS than the ch/ch line. The 14CoS/14CoS cells are two to four times more resistant to menadione killing than ch/ch cells. At concentrations of dicumarol that completely block NMO1 and GSTA1 activities, the 14CoS/14CoS cells show more than twice as much resistance to menadione toxicity than the ch/ch cells. Although superoxide formation is three times higher in untreated 14CoS/14CoS than ch/ch cells, menadione-induced superoxide formation is greater in the dying ch/ch than in the 14CoS/14CoS cells. Cellular resistance to menadione toxicity is correlated with intracellular GSH levels, rather than with the percentage of oxidized glutathione; cytotoxicity is not observed as long as GSH concentrations are sufficiently high (about 5-8 nmol/mg protein). For menadione, the results are consistent with a dominant role of GSH depletion in mediating toxicity and support a protective role for NMO1 activity. This report demonstrates the usefulness of these cell lines as a model system to study mechanisms of oxidative chemically induced toxicity, as well as to understand how intracellular levels of GSH are regulated.

Interactions of mitochondrial inhibitors with methylmercury on spontaneous quantal release of acetylcholine.

The interaction of methylmercury (MeHg) with various inhibitors of mitochondrial function (dinitrophenol, 50 microM; dicoumarol, 100 microM; valinomycin, 20 microM; and ruthenium red, 20 microM) on spontaneous quantal release of acetylcholine was tested at the neuromuscular junction of the rat. The objective was to determine whether these mitochondrial inhibitors blocked the MeHg-induced increase of spontaneous release of acetylcholine, an effect measured electrophysiologically as increased miniature endplate potential (MEPP) frequency. MEPPs were recorded from myofibers of the rat hemidiaphragm using conventional, intracellular microelectrode recording techniques. When given alone, all four inhibitors increased MEPP frequency from resting levels of 1-2/sec (Hz) to approximately 10-60 Hz after a latency which ranged from 5 to 30 min. MEPP frequency subsequently returned to control levels. Subsequent concomitant application of MeHg (100 microM) with dinitrophenol, dicoumarol, or valinomycin increased MEPP frequency sharply to peak values of 40-60 Hz after 15-20 min. MEPP frequency subsided to pre-MeHg levels 10 min later. The time course and peak MEPP frequency elicited by MeHg after pretreatment with these uncouplers were similar to results obtained in preparations treated with MeHg alone. Ruthenium red, a putative specific inhibitor of the Ca2+ uptake uniporter in mitochondria, increased MEPP frequency to 12 Hz after 8.5 min when given alone. MEPP frequency returned to control levels approximately 10 min later. Subsequent application of MeHg and ruthenium red for up to 80 min failed to increase MEPP frequency. The inability of MeHg to increase MEPP frequency in ruthenium red-treated preparations was not due to depletion of acetylcholine nor to block of postjunctional receptors by ruthenium red since subsequent treatment with La3+ (2 mM) increased MEPP frequency to 12.5 Hz within 10 min. Thus, ruthenium red blocked the stimulatory effect of MeHg on MEPP frequency while uncouplers of oxidative phosphorylation and a K+ ionophore did not. The results with ruthenium red are consistent with the proposal that MeHg may block mitochondrial uptake of Ca2+ or promote its release, leading to an increased free cytosolic Ca2+ concentration which in turn stimulates spontaneous release of acetylcholine.

[Dicoumarol as the cause of abortion in mink (author's transl)]. / Dikumarols abortframkallande effekt hos mink

PIP: The use of discoumarol for causing abortion in mink is discussed. D icoumarol affects the coagulation mechanism of the blood. Coumarol derivatives are very similar to the Vitamin-K complex and are assimilated into the enzyme system in the place of Vitamin-K, inactivating the enzyme system. Depending on dosage, dicoumarol can serve as a poison, an anticoagulant, or a means of inducing abortion. In an experiment involving 15 mink, it was found that .2-.6 mg. of dicoumarol/kg of body weight, given through an 8-10 day period resulted in abortion. Doses of 1.2-1.3 mg/kg of body weight given through a 4-6 day period can cause fatal hemorrhaging.^ieng