A new approach to measuring estrogen exposure and metabolism in epidemiologic studies.

Endogenous estrogen plays an integral role in the etiology of breast and endometrial cancer, and conceivably ovarian cancer. However, the underlying mechanisms and the importance of patterns of estrogen metabolism and specific estrogen metabolites have not been adequately explored. Long-standing hypotheses, derived from laboratory experiments, have not been tested in epidemiologic research because of the lack of robust, rapid, accurate measurement techniques appropriate for large-scale studies. We have developed a stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS(2)) method that can measure concurrently all 15 estrogens and estrogen metabolites (EM) in urine and serum with high sensitivity (level of detection=2.5-3.0fmol EM/mL serum), specificity, accuracy, and precision [laboratory coefficients of variation (CV's) < or =5% for nearly all EM]. The assay requires only extraction, a single chemical derivatization, and less than 0.5mL of serum or urine. By incorporating enzymatic hydrolysis, the assay measures total (glucuronidated+sulfated+unconjugated) EM. If the hydrolysis step is omitted, the assay measures unconjugated EM. Interindividual differences in urinary EM concentrations (pg/mL creatinine), which reflect total EM production, were consistently large, with a range of 10-100-fold for nearly all EM in premenopausal and postmenopausal women and men. Correlational analyses indicated that urinary estrone and estradiol, the most commonly measured EM, do not accurately represent levels of total urinary EM or of the other EM. In serum, all 15 EM were detected as conjugates, but only 5 were detected in unconjugated form. When we compared our assay methods with indirect radioimmunoassays for estrone, estradiol, and estriol and enzyme-linked immunosorbent assays for 2-hydroxyestrone and 16alpha-hydroxyestrone, ranking of individuals agreed well for premenopausal women [Spearman r (r(s))=0.8-0.9], but only moderately for postmenopausal women (r(s)=0.4-0.8). Our absolute readings were consistently lower, especially at the low concentrations characteristic of postmenopausal women, possibly because of improved specificity. We are currently applying our EM measurement techniques in several epidemiologic studies of premenopausal and postmenopausal breast cancer.

Nitric oxide prodrug JS-K inhibits ubiquitin E1 and kills tumor cells retaining wild-type p53.

Nitric oxide (NO) is a major effector molecule in cancer prevention. A number of studies have shown that NO prodrug JS-K (O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate) induces apoptotic cell death in vitro and in vivo, indicating that it is a promising new therapeutic for cancer. However, the mechanism of its tumor-killing activity remains unclear. Ubiquitin plays an important role in the regulation of tumorigenesis and cell apoptosis. Our earlier report has shown that inactivation of the ubiquitin system through blocking E1 (ubiquitin-activating enzyme) activity preferentially induces apoptosis in p53-expressing transformed cells. As E1 has an active cysteine residue that could potentially interact with NO, we hypothesized that JS-K could inactivate E1 activity. E1 activity was evaluated by detecting ubiquitin-E1 conjugates through immunoblotting. JS-K strikingly inhibits the ubiquitin-E1 thioester formation in cells in a dose-dependent manner with an IC(50) of approximately 2 microM, whereas a JS-K analog that cannot release NO did not affect these levels in cells. Moreover, JS-K decreases total ubiquitylated proteins and increases p53 levels, which is mainly regulated by ubiquitin and proteasomal degradation. Furthermore, JS-K preferentially induces cell apoptosis in p53-expressing transformed cells. These findings indicate that JS-K inhibits E1 activity and kills transformed cells harboring wild-type p53.

Plasma pharmacokinetics of a liver-selective nitric oxide-donating diazeniumdiolate in the male C57BL/6 mouse.

1. The single-dose plasma pharmacokinetics of O(2)-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) following intravenous (i.v.) and intraperitoneal (i.p.) bolus administration to the male C57BL/6 mouse was studied in an effort to characterize the disposition of the agent and to serve as a basis for the design of in vivo efficacy studies. 2. Plasma V-PYRRO/NO concentrations declined rapidly in a bi-exponential manner after i.v. administration of 5 mg kg(-1) body weight to mouse. The terminal half-life was 9.4 min and the mean residence time was 3.4 min. 3. V-PYRRO/NO was absorbed rapidly following i.p. administration, with peak plasma concentrations being observed 3 min after injection. Levels then declined with a terminal half-life of 11.7 min. The bioavailable fraction from the i.p. compartment was 19%, indicating a high first-pass effect. 4. The results provide additional evidence for a liver-selective metabolism of this nitric oxide-donating prodrug.

Protection of human keratinocyte mtDNA by low-level nitric oxide.

This study was designed to evaluate the DNA damaging effects of nitric oxide and to determine whether the endogenous generation of nitric oxide at low levels in the cell exerts a protective effect against this damage. Damage to mitochondrial and nuclear DNA in normal human epidermal keratinocytes (NHEK) was assessed after treatment of these cells with varying concentrations of S-nitroso-N-acetylpenicillamine, which decomposes to release nitric oxide. The results showed that mitochondrial DNA was more vulnerable to nitric oxide-induced damage than was a similarly sized fragment of the beta-globin gene. To evaluate the effects on DNA damage by pretreatment of cells with low-levels of nitric oxide, NHEK cells were treated with the prodrug V-PYRRO/NO. This agent is metabolized inside these cells and releases small quantities of nitric oxide. The cells then were exposed to damaging amounts of nitric oxide produced by S-nitroso-N-acetylpenicillamine. The results of these studies showed that pretreatment of NHEK cells with V-PYRRO/NO attenuated the mtDNA damage and loss of cell viability produced by exposure to S-nitroso-N-acetylpenicillamine.

Comparison of responses to novel nitric oxide donors in the feline pulmonary vascular bed.

Pulmonary vascular responses to the novel diazeniumdiolate nitric oxide (NO) donors diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt, were investigated and compared in the intact-chest cat. Under conditions of controlled blood flow, when tone in the pulmonary vascular bed had been raised to a high steady level, intralobar injections of diethylamine/NO (0.3-10 microg), diethylenetriamine/NO (10-30 microg), spermine/NO (10-30 microg), sulfite/NO (10-30 microg), and angeli's salt (10-30 microg) caused dose-related decreases in lobar arterial pressure without changing left atrial pressure. In terms of relative vasodilator activity in the pulmonary vascular bed, the dose of the compounds that decreased lobar arterial pressure 4 mm Hg (ED(4) mm Hg) was significantly lower for diethylamine/NO compared to S-nitroso-N-acetylpenicillamine which was significantly less than diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt. The half-life of the vasodilator responses, as measured by 50% response recovery time, to diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt was similar for doses with similar magnitudes of vasodilation, while the half-life to S-nitroso-N-acetylpenicillamine was significantly less than the diazeniumdiolate NO donors. The present data demonstrate that the diazeniumdiolate NO donors diethylamine/NO, diethylenetriamine/NO, spermine/NO, sulfite/NO, and angeli's salt have potent but relatively short-lasting vasodilator activity in the pulmonary vascular bed of the cat.

Chemistry of the diazeniumdiolates. I. Structural and spectral characteristics of the [N(O)NO]- functional group.

Ions of structure X[N(O)NO]-, examples of which have seen increasing use as probes for studying the biology of nitric oxide (NO) over the past decade, have a varied chemical history spanning nearly two centuries. Nevertheless, they have not been widely appreciated for their physicochemical similarities. Here we begin a series of systematic inquiries into the fundamental chemistry of such compounds aimed at identifying both the characteristics that justify considering them as a group and the factors that contribute to observed differences in their physicochemical properties. In the present paper, X-ray structures in which X is SO3- (1), O- (2), Ph (3), and Et2N (5), as well as that of the gem-disubstituted carbon derivative CH2[N(O)NO]2-(2) (4), are compared. All their O-N-N-O systems are essentially planar, with cis oxygens and an N-N linkage exhibiting considerable double-bond character. The ultraviolet spectrum of the isolated chromophore consists of a relatively intense ( approximately 6-10 mM(-1) x cm(-1) per [N(O)NO]- group) absorption at 248-250 nm (for 2 and 5) that is red shifted by through-space Stark interactions (e.g., by approximately 10 nm in 1 and 4) as well as by conjugative interaction with X (lambda(max) = 284 nm for 3). Infrared and Raman spectra for the widely used pharmacological probe 5 were determined, with analysis of vibrational modes being aided by comparison with the spectra of the [15N(O)15NO]- isotopomer and density functional theory calculations at the B3LYP/6-311++G** level. To address confusion that has arisen in the literature resulting from rather widespread use of differing trivial designations for this class of compounds, a unifying nomenclature system is recommended in which compounds containing the [N(O)NO]- moiety are named as diazeniumdiolates. It is hoped that these and other efforts to understand and predict the physicochemical similarities and differences among different members of the diazeniumdiolate class will aid in reaping their full potential in the area of rational drug design.

L-tyrosine and nitric oxide synergize to prevent cytotoxic effects of superoxide.

We found previously that the nitric oxide donor DEA/NO enhanced lipid peroxidation, DNA fragmentation, and cytotoxicity in human bronchial epithelial cells (BEAS-2B) when they were cultured in LHC-8 medium containing the superoxide-generating system hypoxanthine/xanthine oxidase (HX/XO). We have now discovered that DEA/NO's prooxidant action can be reversed by raising the L-tyrosine concentration from 30 to 400 microM. DEA/NO also protected the cells when they were cultured in Dulbecco's Modified Eagle's Medium (DMEM), whose standard concentration of L-tyrosine is 400 microM. Similar trends were seen with the colon adenoma cell line CaCo-2. Since HPLC analysis of cell-free DMEM or LHC-8 containing 400 microM L-tyrosine, DEA/NO, and HX/XO revealed no evidence of L-tyrosine nitration, our data suggest the existence of an as-yet uncharacterized mechanism by which L-tyrosine can influence the biochemical and toxicological effects of reactive nitrogen species.

Analysis of vasodilator responses to novel nitric oxide donors in the hindquarters vascular bed of the cat.

Controlled release of nitric oxide (NO*) may be useful in the treatment of a variety of vascular disorders. NO* donors of the diazeniumdiolate family with different rates of spontaneous NO* release have been synthesized. In the current study responses to seven diazeniumdiolate NO* donors (DEA/NO*, DETA/NO*, OXI/NO*, PIPERAZI/NO*, PROLI/NO*, SPER/NO*, and SULFI/NO*) were investigated in the hindquarters vascular bed of the cat. Intravenous injections of all NO* donors caused dose-dependent decreases in systemic arterial pressure and the rank order of potency was SNP > DEA/NO* > PIPERAZI/NO* > SPER/NO* > PROLI/NO* > OXI/NO*. Injections of all NO* donors into the hindlimb perfusion circuit caused dose-related decreases in hindquarters perfusion pressure that were similar to the order of potency in decreasing systemic arterial pressure. The rank order of the time required for the response to return to 50% of the maximal decrease in pressure (T(1/2)) and total duration of action of the NO* donors was SPER/NO* > PIPERAZI/NO* > DEA/NO* > OXI/NO* > DETA/NO* > PROLI/NO* > SULFI/NO*. After treatment with the NO* synthase inhibitor, N(omega)-nitro-L-arginine methyl ester (100 mg/kg, i.v.), hindlimb vasodilator responses to the NO* donors were not significantly different, but vasodilator responses to acetylcholine were significantly reduced. After treatment with zaprinast (2 mg/kg, i.v.), a type V cyclic 3',5'-guanosine monophosphate-specific phosphodiesterase inhibitor, the duration of vasodilator responses to the NO* donors, as measured by T(1/2), was increased significantly, whereas the duration of the response to the beta2-adrenergic receptor agonist albuterol was unchanged. These data suggest that diazeniumdiolate NO* donors are endothelium-independent, directly stimulate soluble guanylate cyclase, and decrease vascular resistance by increasing cyclic 3',5'-guanosine monophosphate levels in the hindquarters vascular bed of the cat.

Overexpression of glutathione S-transferase II and multidrug resistance transport proteins is associated with acquired tolerance to inorganic arsenic.

Recent work shows that long-term exposure to low levels of arsenite induces malignant transformation in a rat liver epithelial cell line. Importantly, these chronic arsenic-exposed (CAsE) cells also develop self-tolerance to acute arsenic exposure. Tolerance is accompanied by reduced cellular arsenic accumulation, suggesting a mechanistic basis for reduced arsenic sensitivity. The present study examined the role of xenobiotic export pumps in acquired arsenic tolerance. Microarray analysis of CAsE cells showed increased expression of the genes encoding for glutathione S-transferase Pi (GST-Pi), multidrug resistance-associated protein genes (MRP1/MRP2, which encode for the efflux transporter Mrp1/Mrp2) and the multidrug resistance gene (MDR1, which encodes for the efflux transporter P-glycoprotein). These findings were confirmed at the transcription level by reverse transcription-polymerase chain reaction and at the translation level by Western-blot analysis. Acquired arsenic tolerance was abolished when cells were exposed to ethacrynic acid (an inhibitor of GST-Pi), buthionine sulfoximine (a glutathione synthesis inhibitor), MK571 (a specific inhibitor for Mrps), and PSC833 (a specific inhibitor for P-glycoprotein) in dose-dependent fashions. MK571, PSC833, and buthionine sulfoximine markedly increased cellular arsenic accumulation. Consistent with a role for multidrug resistance efflux pumps in arsenic resistance, CAsE cells were found to be cross-resistant to cytotoxicity of several anticancer drugs, such as vinblastine, doxorubicin, actinomycin-D, and cisplatin, that are also substrates for Mrps and P-glycoprotein. Thus, acquired tolerance to arsenic is associated with increased expression GST-Pi, Mrp1/Mrp2 and P-glycoprotein, which function together to reduce cellular arsenic accumulation.

Chemistry of the diazeniumdiolates. 3. Photoreactivity.

We have found O(2)-substituted diazeniumdiolates, compounds of structure R(2)N-N(O)=NOR' that are under development for various possible pharmaceutical uses, to be rather photosensitive. With R = ethyl and R' = methyl, benzyl, or 2-nitrobenzyl, the observed product distributions suggest that two primary pathways are operative. A minor pathway involves the extrusion of nitrous oxide (N(2)O) with simultaneous generation of R(2)N(*) and R'O(*), which may then form amines, aldehydes, and alcohols. The major reaction pathway is an interesting photochemical cleavage of the N=N bond to form a nitrosamine (R(2)NN=O) and an oxygen-substituted nitrene (R'ON). The intermediacy of the O-nitrene was inferred from the production of abundant oxime, via rearrangement of the O-nitrene to a C-nitroso compound (R'ON --> O=NR'), and subsequent tautomerization to the more stable oxime. Involvement of the O-nitrene was confirmed by trapping with 2,3-dimethyl-2-butene to form the aziridine and with oxygen to generate the nitrate ester. 2-Nitro substitution on the benzyl derivative had surprisingly little effect on the reaction course. For each compound examined, minor amounts of nitric oxide (NO), presumably produced by secondary photolysis of the nitrosamine, were observed. Time-resolved infrared experiments provided additional support for the above reaction pathways and confirmed that the nitrosamine is a primary photoproduct. We have also found that the relative contributions of the reaction pathways can be altered in certain derivatives. For example, when R' = 2,4-dinitrophenyl, the contribution of the nitrosamine/O-nitrene-forming pathway was diminished. Pharmacological implications of these results are discussed.

Chemistry of the diazeniumdiolates. 2. Kinetics and mechanism of dissociation to nitric oxide in aqueous solution.

Diazeniumdiolate ions of structure R(2)N[N(O)NO](-) (1) are of pharmacological interest because they spontaneously generate the natural bioregulatory species, nitric oxide (NO), when dissolved in aqueous media. Here we report the kinetic details for four representative reactivity patterns: (a) straightforward dissociation of the otherwise unfunctionalized diethylamine derivative 2 (anion 1, where R = Et) to diethylamine and NO; (b) results for the zwitterionic piperazin-1-yl analogue 4, for which the protonation state of the neighboring basic amine site is an important determinant of dissociation rate; (c) data for 5, a diazeniumdiolate derived from the polyamine spermine, whose complex rate equation can include terms for a variety of medium effects; and (d) the outcome for triamine 6 (R = CH(2)CH(2)NH(3)(+)), the most stable structure 1 ion identified to date. All of these dissociations are acid-catalyzed, with equilibrium protonation of the substrate preceding release of NO. Specific rate constants and pK(a) values for 2-6 have been determined from pH/rate profiles. Additionally, a hypsochromic shift (from approximately 250 to approximately 230 nm) was observed on acidifying these ions, allowing determination of a separate pK(a) for each substrate. For 6, the pK(a) value obtained kinetically was 2-3 pK(a) units higher than the value obtained from the spectral shift. Comparison of the ultraviolet spectra for 6 at various pH values with those for O- and N-alkylated diazeniumdiolates suggests that protonation at the R(2)N nitrogen initiates dissociation to NO at physiological pH, with a second protonation (at oxygen) accounting for both the spectral change and the enhanced dissociation rate at pH <4. Our results help to explain the previously noted variability in dissociation rate of 5, whose half-life we found to increase by an order of magnitude when its concentration was raised from near-zero to 1 mM, and provide mechanistic insight into the factors that govern dissociation rates among diazeniumdiolates of importance as pharmacologic progenitors of NO.

Nitric oxide induces metallothionein (MT) gene expression apparently by displacing zinc bound to MT.

The metal binding protein metallothionein (MT) is involved in zinc homeostasis since it typically binds large amounts of zinc. Free zinc can control MT gene expression by interacting with metal-sensitive transcription factors. However, the precise factors governing intracellular release of metal ions from MT remain unknown. Aerobic nitric oxide (NO) can nitrosate thiol groups in proteins, and MT-bound cadmium is released by NO exposure. Thus, we hypothesized that NO may also be effective at displacing zinc from MT in cultured cells and that this could be an important physiological control mechanism in zinc homeostasis and utilization. In this study, DETA/NO, an agent that spontaneously generates NO with a 20-h half life in physiological media, was used to study the release of zinc from MT and the induction of MT in TRL1215 cells (a normal rat liver cell line). Zinc or cadmium was given at levels inducing MT production, followed by DETA/NO (20-200 microM) to produce controlled NO exposure in both cell lines. Although both metals activated MT gene expression, MT-I mRNA and MT protein were further increased when DETA/NO was given after zinc or cadmium treatment. Additionally, NO from DETA/NO clearly displaced MT-bound zinc, as evidenced by G-75 gel-filtration chromatography. The released zinc or cadmium probably then stimulates further MT gene expression. These results suggest that NO may play an important role in regulation of cellular zinc homeostasis by providing a controlled release mechanism for metal ions stored in MT, and NO-mediated release of MT-bound zinc could in turn activate gene expression, such as with the MT gene.

The secondary amine/nitric oxide complex ion R(2)N[N(O)NO](-) as nucleophile and leaving group in S9N)Ar reactions.

Ions of structure R(2)N[N(O)NO](-) and their alkylation products have seen increasing use as nitric oxide (NO)-generating agents for biomedical research applications. Here we show that such diazeniumdiolate anions can readily displace halide from a variety of electrophilic aza- or nitroaromatic substrates to form O(2)-arylated derivatives of structure R(2)N-N(O)=N-OAr. The site of arylation and the cis arrangement of the oxygens were confirmed by X-ray crystallography. Displacement by various nucleophiles showed R(2)N[N(O)NO](-) to be a reasonably good leaving group, with rate constants for displacement by hydroxide, methoxide, and isopropylamine that were between those of chloride and fluoride in the S(N)Ar reactions we surveyed. The Meisenheimer intermediate could be spectrally observed. These O(2)-aryl diazeniumdiolates proved capable of reacting with the nucleophilic sulfur of the HIV-1 p7 nucleocapsid protein's zinc finger assembly to eject the zinc, disrupting a structural motif critical to viral replication and suggesting possible utility in the drug discovery realm.

Inhibition of oxidized low-density lipoprotein-induced apoptosis in endothelial cells by nitric oxide. Peroxyl radical scavenging as an antiapoptotic mechanism.

Proatherogenic oxidized low-density lipoprotein (oxLDL) induces endothelial apoptosis. We investigated the anti-apoptotic effects of intracellular and extracellular nitric oxide (*NO) donors, iron chelators, cell-permeable superoxide dismutase (SOD), glutathione peroxidase mimetics, and nitrone spin traps. Peroxynitrite (ONOO-)-modified oxLDL induced endothelial apoptosis was measured by DNA fragmentation, TUNEL assay, and caspase-3 activation. Results indicated the following: (i) the lipid fraction of oxLDL was primarily responsible for endothelial apoptosis. (ii) Endothelial apoptosis was potently inhibited by *NO donors and lipophilic phenolic antioxidants. OxLDL severely depleted Bcl-2 levels in endothelial cells and *NO donors restored Bcl-2 protein in oxLDL-treated cells. (iii) The pretreatment of a lipid fraction derived from oxLDL with sodium borohydride or potassium iodide completely abrogated apoptosis in endothelial cells, suggesting that lipid hydroperoxides induce apoptosis. (iv) Metalloporphyrins dramatically inhibited oxLDL-induced apoptosis in endothelial cells. Neither S-nitrosation of caspase-3 nor induction of Hsp70 appeared to play a significant role in the antiapoptotic mechanism of *NO in oxLDL-induced endothelial apoptosis. We propose that cellular lipid peroxyl radicals or lipid hydroperoxides induce an apoptotic signaling cascade in endothelial cells exposed to oxLDL, and that *NO inhibits apoptosis by scavenging cellular lipid peroxyl radicals.

V-PYRRO/NO: an hepato-selective nitric oxide donor improves porcine liver hemodynamics and function after ischemia reperfusion.

BACKGROUND: The role of nitric oxide (NO) in ischemia reperfusion (I/R) injury is controversial as both beneficial and harmful effects have been reported. We explored the potential role of a pharmacological agent recently shown to generate NO metabolically in the liver in an animal model of transplantation. METHODS: The effect of a selective hepatic NO donor, O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), on hepatic hemodynamics and biliary function was evaluated in both the in situ and I/R pig liver. RESULTS: V-PYRRO/NO significantly reduced in situ hepatic vascular resistance (HVR) without altering systolic blood pressure. Portal vein flow was essentially unchanged during in situ infusions while hepatic artery flow nearly doubled (P=0.03). After I/R, V-PYRRO/NO infusions significantly reduced both portal vein pressure (PVP) and HVR (P=0.04). Also, serum bile acid clearance increased from 15% when taurocholate (TC) was infused alone to 46% (P=0.007) when infused simultaneously with V-PYRRO/NO. Aqueous bile production tripled with TC and V-PYRRO/NO as compared to TC alone (P=0.04). Analysis of bile outputs revealed a significant increase in biliary cholesterol, biliary phospholipid, and biliary bile acid (P<0.05) with V-PYRRO/NO infusion. CONCLUSIONS: The hepato-selective nitric oxide donor, V-PYRRO/NO, reduced hepatic resistance parameters of the pig liver both before and after I/R and improved the plasma clearance of bile acid and biliary outputs of bile acid-dependent compounds. The augmented function observed after I/R may be due to improvements in hepatic blood flow secondary to altered hepatic hemodynamics.

Pathophysiology of a sickle cell trait mouse model: human alpha(beta)(S) transgenes with one mouse beta-globin allele.

As a potential model for sickle cell trait (AS), we examined mice containing one normal mouse beta-globin allele in combination with a human hemoglobin S (h(alpha)beta(S)) transgene (m(beta)/hS). The mice segregated into two subpopulations containing low and high proportions of hemoglobin S (m(beta)/hS1 and m(beta)/hS2, respectively) that was associated with one or two human h(alpha)beta(S) transgenes. We noted striking kidney pathology (cortical cysts, hyperplastic tubules, and glomerulonephritis), increasing with age and with greater severity in m(beta)/hS1. mBeta/hS2 animals were largely tolerant to 5% O(2) for 1 h, whereas 80% of m(beta)/hS1 mice died, exhibiting acute sequestration of erythrocytes in spleen, liver, and heart. These pathologies appear to result from a decreased oxygen affinity of the hybrid (human alpha/mouse beta) hemoglobins with a mild beta-thalassemia phenotype. Thus, these mouse models of sickle trait seem to manifest their renal pathology and sensitivity to hypoxia by mechanisms related to low tissue oxygen delivery and are different from the human syndrome. Analyses of parameters such as P(50), red cell indices, and genetic background are necessary in establishing potential relevance of any mouse model of the sickle cell syndromes.

Reaction of nitric oxide at the beta-carbon of enamines. A new method of preparing compounds containing the diazeniumdiolate functional group.

The reaction of nitric oxide (NO) with enamines has been investigated. Unlike previously reported reactions of NO as a free radical with alkenes, the electrophilic addition of NO to the beta-carbon of enamines results in the formation of compounds containing the diazeniumdiolate functional group (-[N(O)NO](-)). This reaction between NO and enamines has been shown to be quite general and a variety of enamine-derived diazeniumdiolates have been isolated and characterized. While enamines derived from aldehydes and ketones whose structures allow for sequential multiple electrophilic additions tended to undergo overreaction leading to unstable products, it has been shown that this complication may be overcome by suitable choice of reaction solvent. The products obtained may exist as zwitterionic iminium salts or as neutral species depending upon the structure of the parent enamine. The diazeniumdiolate derived from 1-(N-morpholino)cyclohexene is unique among the new compounds in that it spontaneously releases NO upon dissolution in buffered aqueous solution at pH 7.4 and 37 degrees C. While the total quantity of NO released by this material (ca. 7% of the theoretical 2 moles) is apparently limited by a competing reaction in which it hydrolyzes to an alpha-diazeniumdiolated carbonyl compound and the parent amine, this feature may prove to be of great value in the development of multiaction pharmaceuticals based upon this new type of NO-releasing compound. Reports of enzymatic (oxidative) release of NO from previously known carbon-bound diazeniumdiolates also suggest that analogues of these compounds may be useful as pharmaceutical agents. This new method of introducing the relatively rarely studied diazeniumdiolate functional group into organic compounds should lead to further research into its chemical and biological properties.

Diazeniumdiolates: pro- and antioxidant applications of the "NONOates".

Diazeniumdiolates are compounds containing the X-[N(O)NO](-) structural unit that as a class offer many advantages as tools for probing the roles of nitric oxide (NO) in biological redox processes. Available examples in which X is a secondary amine group spontaneously generate up to two molecules of NO per [N(O)NO](-) unit when dissolved in aqueous media; their half-lives range from 2 s (for X = L-prolyl) to 20 h [for X = (H(2)NCH(2)CH(2))(2)N] at pH 7. 4 and 37 degrees C, and are in general relatively little influenced by medium effects or metabolism. When X = O(-) (Angeli's salt), first-order dissociation produces NO(-) rather than NO, but the ion becomes an NO source on 1-electron oxidation; diazeniumdiolate-derived NO can also be used to generate reactive nitrogen/oxygen species with higher nitrogen oxidation states (+3 and +4) in the presence of selected oxidizing agents. The advantages of diazeniumdiolates in biomedical research are briefly illustrated with examples from the recent literature probing NO's role in inhibiting oxidative drug metabolism, radical-induced lipid oxidation, the cytotoxicity of reactive oxygen species, and ischemia-induced vascular reoxygenation injury. Future work with this compound class should provide further insight into the mechanisms of NO's involvement in pro- and antioxidant processes, and may well lead to important medicinal advances, including reversal of cerebral vasospasm and radiosensitization of hypoxic tumors.

Induction of proliferative lesions of the uterus, testes, and liver in swiss mice given repeated injections of sodium arsenate: possible estrogenic mode of action.

Inorganic arsenic (As) is a human carcinogen but has not been unequivocally proven carcinogenic in rodents. For instance, one older study indicates that repeated iv injections of sodium arsenate might induce lymphomas in Swiss mice (58% incidence) (Osswald and Goerttler, Verh. Dtsch. Ges. Pathol. 55, 289-293, 1971), but it was considered inadequate for critical evaluation of carcinogenic potential largely because of issues in experimental design. Therefore, we studied repeated iv sodium arsenate injection and neoplastic response in male and female Swiss mice. Groups (n = 25) of mice received sodium arsenate (0.5 mg/kg, iv) or saline (control) once/week for 20 weeks and were observed for a total of 96 weeks when the study ended. Differences in survival and body weights were unremarkable. In females, arsenate induced marked increases in the incidence and severity of cystic hyperplasia of the uterus compared against controls. Arsenate also was associated with a rare adenocarcinoma of the uterus. Hyperplastic uterine epithelium from arsenate-exposed animals showed strong positive immunostaining for the proliferating cell nuclear antigen (PCNA). There was also an upregulation of estrogen receptor (ER) immunoreactive protein in the early lesions of uterine luminal and glandular hyperplasia, although a progressive decrease in its expression was seen in the severe hyperplastic or neoplastic epithelium. In common with the preneoplastic and neoplastic gynecological lesions in humans, the levels of immunoreactive inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine-containing proteins were greater in the uterine hyperplastic epidermis and their intensity was positively correlated with the severity of the lesions. Arsenate-induced uterine hyperplastic lesions also showed a strong upregulation of cyclin D1, an estrogen-associated gene product essential for progression through the G1 phase of the cell cycle. In other tissues, arsenate increased testicular interstitial cell hyperplasia incidence and severity over control but without affecting the incidence of tubular degeneration. Arsenate also induced increases in hepatic proliferative lesions (HPL; foci of alteration + neoplasia), but only in females. Significant skin changes (incidence of hyperkeratotic lesions) and renal lesions (severity of nephropathy) also occurred in arsenate-treated females. Thus, repeated arsenate exposure, though not outright tumorigenic in the present study, was associated with proliferative, preneoplastic lesions of the uterus, testes, and liver. Estrogen treatment has been associated with proliferative lesions and tumors of the uterus, female liver, and testes in other studies, supporting a hypothesis that arsenate might somehow act through an estrogenic mode of action.

Conversion of a polysaccharide to nitric oxide-releasing form. Dual-mechanism anticoagulant activity of diazeniumdiolated heparin.

We describe heparin/diazeniumdiolate conjugates that generate nitric oxide (NO) at physiological pH. Like the heparin from which they were prepared, they inhibit thrombin-induced blood coagulation. Unlike heparin, they can also inhibit and reverse ADP-induced platelet aggregation (as expected for an NO-releasing agent), suggesting potential utility as dual-action antithrombotics.