N-nitrosation by nitrite ion in neutral and basic medium.

Formaldehyde catalyzed the conversion of various secondary amines to nitrosamines in the pH range 6.4 to 11.0. Chloral was also an effective catalyst. The reaction proceeds easily enough to have potential synthetic applications; the proposed mechanism could explain some reported anomalies regarding the synthesis of carcinogenic N-nitroso compounds in vivo and in vitro.

Nitric oxide suppression of human hematopoiesis in vitro. Contribution to inhibitory action of interferon-gamma and tumor necrosis factor-alpha.

IFN-gamma and TNF-alpha, potent inhibitors of hematopoiesis, induce nitric oxide synthase (NOS) in various cell types. When normal human bone marrow (BM) or CD34+ cells were exposed to NO, inhibition of colony formation was dose dependent and direct. NO induced apoptosis in BM progenitors, as shown by electrophoretic detection of DNA degradation and deoxynucleotidyl transferase assay. Using PCR and immunoprecipitation, we found inducible NOS (iNOS) mRNA and iNOS protein in BM after stimulation with IFN-gamma or TNF-alpha. iNOS mRNA was also detected by PCR in highly purified CD34+ cells; TNF-alpha or IFN-gamma increased iNOS expression. The presence of iNOS in CD34+ cells was confirmed in single cells by immunochemical staining. NG-Monomethyl-L-arginine (MM-Arg), an NOS inhibitor, partially reversed the effects of TNF-alpha and, to a lesser extent, IFN-gamma in methylcellulose culture of total BM and CD34+ cells, and inhibited apoptosis of BM cells induced by these cytokines. When the effects of competitive iNOS inhibition were tested on more immature progenitors, MM-Arg increased the number of long-term BM culture-initiating cells in control cultures but failed to protect these cells from the inhibitory action of IFN-gamma and TNF-alpha. Our results suggest that NO may be one mediator of cytokine-induced hematopoietic suppression.

Reduction of rat liver carcinogenicity of 4-nitrosomorpholine by alpha-deuterium substitution.

Groups of 30 males Sprague-Dawley rats were given 4-nitrosomorpholine-3,3,5,5-d in their drinking water at concentrations of 0.35 and 0.07 X 10(-3) M for 30 weeks. Two similar groups of rats were simultaneously given unlabeled 4-nitrosomorpholine (NM) at the same malar concentrations; all animals were observed throughout their lives. Those receiving the alpha-deuterium-labeled compound had significantly fewer liver tumors than did the corresponding animals receiving the unlabeled compound. The difference in potency appeared to be at least fivefold, a magnitude consistent with a primary kinetic isotope effect on the carcinogenic action of NM. Thus breakage of a bond linking a hydrogen (deuterium) atom with carbon adjacent to the nitrosamino function may be involved in a rate-limiting step of carcinogenesis by NM.

Carcinogen chemistry. I. Reactions of protonated dialkylnitrosamines leading to alkylating and aminoalkylating agents of potential metabolic significance.

Three distinct modes of protolytic dialkylnitrosamine fragmentation were observed when we followed the time dependence of the nuclear magnetic resonance (NMR) spectra of seven nitrosamines in superacid solution: 1) In equimolar HSO3F: SbF5 ("magic acid"), dimethylnitrosamine was cleaved to the protonated Schiff base of formaldehyde and methylamine, and diethylnitrosamine was similarly converted to the protonated acetaldehydeethylamine Schiff base.2) By contrast, of the five dipropyl-and dibutylnitrosamines were studied, all cleaved nonoxidatively under these conditions (with loss of nitrogen gas) to the corresponding propyl or butyl cations. The carbocations thus produced underwent condensation and fragmentation to form the tert-butyl cation as the principal product ultimately observable by NMR. 3) Thethird fragmentation mechanism, which involved denitrosation to the dialkylammonium ion, was observed only as a minor pathway in the sulfuric or fluorosulfuric acid protolysis of dimethylnitrosamine. The mechanisms that are postulated for these cleavage reactions, if functioning in vivo, could account for several metabolic observations that have proved difficult to reconcile with previous conceptions of nitrosamine metabolism.

Focal suppression and induction of hyperplasia by the bladder carcinogens butyl(4-hydroxybutyl)nitrosamine and buty(3-carboxypropyl)nitrosamine in organ-cultured rat bladder epithelium.

The effects of the bladder carcinogens butyl(4-hydroxybutyl)nitrosamine (BBN) and butyl(3-carboxypropyl)-nitrosamine (BCPN) on proliferating transitional rat epithelium in organ culture were studied. At low to intermediate concentration ranges (0.5--2.9 mM), both compounds appeared to stimulate hyperplasia in some regions of epithelia. The major effect of both carcinogens, however, was to suppress hyperplasia in other regions of epithelia and, at higher concentrations (5--6 mM), to cause necrosis. For comparable concentrations, BBN was more effective in suppressing proliferation and causing necrosis than was BCPN.

Selective induction of intestinal tumors in rats by methyl(acetoxymethyl)nitrosamine, an ester of the presumed reactive metabolite of dimethylnitrosamine.

Methyl(acetoxymethyl)nitrosamine (DMN-OAc) was synthesized and tested for toxicity and carcinogenicity in rats to test the hypothesis that alpha-hydroxylation is required for metabolic activation of dimethylnitrosamine (DMN) to a reactive, proximate carcinogen. The acute median lethal doses (LD50) of DMN-OAc and DMN injected ip into 5-week-old male Sprague-Dawley (Charles River (CD) rats were determined to be 0.19 and 0.59 mmole/kg body weight or 25 mg DMN-OAc/kg and 44 mg DMN/kg body weight, respectively. Single ip injections of one-half the LD50 DMN-OAc (13 mg/kg body weight) in 5-week-old rats of both sexes resulted in a high incidence of epithelial tumors of the intestinal tract. Mean survival times for rats with intestinal tumors were 353 days for males and 433 days for females. Tumors were rarely found at other sites. DMN at equivalent toxic (one-half the LD50, 22 mg/kg) and molar (= one-sixth LD50, 7.0 mg/kg) dose levels, yielded (as expected) tumors of kidneys, lungs, and occasionally other organs, but at a much lower incidence. The finding of the potent carcinogenicity of DMN-OAc supported the postulate that alpha-hydroxylation of DMN in vivo generates a proximate carcinogen.

Concurrent generation of methylamine and nitrite during denitrosation of N-nitrosodimethylamine by rat liver microsomes.

With the goal of identifying the organic amine product(s) of enzymatic N-nitrosodimethylamine (NDMA) denitrosation, 4 mM NDMA was incubated with liver microsomes from ethanol-treated rats. The concentrations of dimethylamine and methylamine were determined by derivatization with 2,4-dinitrofluorobenzene followed by gas chromatography-mass spectrometry. There was no net increase in the concentration of dimethylamine during incubation, but the yield of methylamine was equimolar with that of nitrite. Additional incubations of NDMA using acetone-induced microsomes, 1 mM and 0.1 mM substrate, gave methylamine/nitrite ratios of 0.9 and 0.7, respectively, confirming the quantitative linkage between these two products. Control incubations conducted with pure methylamine or dimethylamine indicated that the secondary amine is not a significant intermediate in the metabolic generation of the primary amine. Experiments with 15N-labeled NDMA showed that the methylamine nitrogen came from the amino moiety of the nitrosamine. The results suggest that NDMA metabolism is best viewed as a competition between at least two important pathways, demethylation and the presumably deactivating denitrosation route, a formulation which seems to account for the previously reported detection of methylamine as a urinary metabolite of NDMA and for the production of less than theoretical yields of labeled dinitrogen gas during NDMA metabolism.

Decontamination and disposal of nitrosoureas and related N-nitroso compounds.

An improved procedure for chemically decontaminating residues of nitrosoureas and related N-nitroso compounds ("nitrosamides") commonly used in the cancer research laboratory is proposed. Treatment of accumulated wastes with aluminum:nickel alloy powder while progressively increasing the basicity of the medium consistently led to at least 99.98% destruction of each nitrosamide tested. Hazardous diazoalkanes were never detected in yields of greater than 0.1%. The mutagenicity of the completed reaction mixtures was never more than 3 times background except when the N-nitroso compound contained a 2-chloroethyl group. In most cases, the completeness of reaction could be determined chromatographically, not only to demonstrate the disappearance of the starting N-nitroso compound, but also to follow production of identifiable products in sufficient abundance to account for the starting material destroyed; none of the organic products observed was mutagenic in any of the four tester strains used. The procedure described herein proved reliable in two checker laboratories besides our own when applied to mixtures of seven N-nitroso compounds: N-methyl-N-nitroso-p-toluene-sulfonamide; N-methyl-N-nitrosourethane; N-methyl-N-nitrosourea; N-methyl-N'-nitro-N-nitrosoguanidine; N-ethyl-N-nitrosourea; N-ethyl-N'-nitro-N-nitrosoguanidine; and N-ethyl-N-nitrosourethane. All of the other procedures investigated for destruction of nitrosamides, including the widely used approach of dissolving the nitrosamides in alkali, were associated with important disadvantages.

Methylation versus ethylation of DNA in target and nontarget tissues of Fischer 344 rats treated with N-nitrosomethylethylamine.

Bioactivation of N-nitrosomethylethylamine can be initiated by hydroxylation of either the methyl or ethyl moiety leading to an ethylating or methylating intermediate, respectively. This study was designed to determine which of these metabolic pathways predominates in vivo and to what extent DNA is alkylated in the target and nontarget tissues. Adult male Fischer 344 rats received a single i.p. or p.o. dose (4.4 mg/kg, 0.05 mmol/kg) of N-nitrosomethylethylamine, 14C-labeled in either the methyl or ethyl group (survival time, 4 h). DNA was analyzed by Sephasorb-HP chromatography following acid hydrolysis in 0.1 M HCl. Concentrations of 7-methylguanine in hepatic DNA were 170-200 times higher than those of 7-ethylguanine. This is approximately 2.6 times the 7-methylguanine:7-ethylguanine ratio of 68, observed when DNA is reacted in vitro with equimolar amounts of the direct alkylating agents N-nitrosomethylurea and N-nitrosoethylurea, suggesting that hydroxylation at the alpha-position of the ethyl group of N-nitrosomethylethylamine proceeds at about 2.6 times the rate as at the methyl group. Concentrations of 7-methylguanine in liver were approximately 15 times higher than in kidney, 100 times higher than in esophagus, and 200 times higher than in lung. Addition of ethanol to the drinking water (5%) caused a slight interorgan shift in metabolism with a decrease in the 7-methylguanine ratio for liver:esophagus by 50% and an increase in the 7-methylguanine ratio for liver:kidney by 40%.

Hypoxic mammalian cell radiosensitization by nitric oxide.

The bioregulatory molecule, nitric oxide (NO), was evaluated as a hypoxic cell radiosensitizer. Authentic NO gas was nearly as effective as oxygen in radiosensitizing hypoxic Chinese hamster V79 lung cells as evaluated using clonogenic assays. When NO was delivered to hypoxic Chinese hamster V79 cells using the NO-releasing agent (C2H5)2N[N(O)-NO]- Na+, radiosensitization was also observed with a sensitizer enhancement ratio of 2.4 (1 mM (C2H5)2N[N(O)NO]-Na+). Aerobic radiosensitivity was not affected at this concentration. The hypoxic cell radiosensitization properties of (C2H5)2N[N(O)NO]-Na+, coupled with the vasodilatory effects of NO on tumor vasculature, suggest that such agents open a new avenue of research in radiation oncology.

Nitric oxide/nucleophile complexes inhibit the in vitro proliferation of A375 melanoma cells via nitric oxide release.

Cell-mediated antitumor effects have, in part, been attributed to the production of NO. Compounds which generate NO might, therefore, be useful in attenuating the growth of tumor cells. Six nitric oxide/nucleophile adducts that release NO spontaneously in solution were tested for their effectiveness in inhibiting DNA synthesis in A375 human melanoma cells. The complexes of NO with spermine, 3-(n-propylamino)propylamine (PAPA/NO), and diethylamine reduced thymidine incorporation by 50% at concentrations of 24, 44, and 128 microM, respectively. The degree of inhibition was, in general, related to the rate and extent of NO release in solution. A melanoma cell clone sensitive to interleukin 1-mediated cytostasis (A375-C6) was no more sensitive to PAPA/NO than a clone resistant to interleukin 1 (A375-C5), suggesting that the differing inhibitory effects of interleukin 1 in the two A375 cell clones are not due to a differential sensitivity to nitric oxide. Oxymyoglobin (125 microM), a known scavenger of NO, restored the ability of A375-C6 cells to incorporate thymidine in the presence of up to 200 microM PAPA/NO. When PAPA/NO was added to a solution of oxymyoglobin, nitrosylmyoglobin was formed, indicating that the protective effect of myoglobin was due to scavenging of NO. The results are consistent with a nitric oxide-mediated mechanism for NO/nucleophile cytostasis and suggest that such compounds may be useful as tools for investigating the role of reactive nitrogen intermediates in cytostasis and cytotoxicity.

Sex differences in the single-dose toxicokinetics of N-nitrosomethyl(2-hydroxyethyl)amine in the rat.

The single-dose toxicokinetics of N-nitrosomethyl(2-hydroxyethyl)amine (NMHA) has been characterized in 8-week-old Fischer 344 rats by analysis using high-performance liquid chromatography of serial blood samples. An i.v. bolus dose of 0.6 mumol/kg to male rats revealed biphasic first-order elimination with a terminal half-life of 37.4 +/- 1.7 min for unchanged NMHA and 101 +/- 6 min for total radioactivity, and extensive conversion to polar metabolites was seen in the high-performance liquid chromatographic assays. The systemic blood clearance and apparent steady-state volume of distribution for unchanged NMHA were 13.1 +/- 0.9 ml/min/kg, and 685 +/- 31 ml/kg, respectively. Renal blood clearance and intrinsic hepatic clearance were estimated to be 0.805 +/- 0.024 and 16.7 +/- 2.1 ml/min/kg, respectively. A similar dose given to female rats yielded a terminal half-life for NMHA of 27.2 +/- 1.2 min, a steady-state volume of distribution of 652 +/- 23 ml/kg, and systemic blood, renal blood, and intrinsic hepatic clearances of 16.9 +/- 1.3, 1.45 +/- 0.14, and 22.5 +/- 0.3 ml/min/kg, respectively. The sex differences in terminal half-life and systemic blood, renal blood, and intrinsic hepatic clearances were significant at the P less than 0.05 level. Larger doses given by gavage, which appeared to be completely absorbed from the gut, indicated systemic bioavailabilities for unchanged NMHA of 78 +/- 10% and 69 +/- 1% for male and female rats, respectively. Binding of NMHA to plasma proteins was found to be negligible. Taken together the data allow for the conclusion that the observed sex differences in toxicokinetic parameters are due to differences in the intrinsic hepatic clearance of the compound. This difference in the ability of the liver to metabolize NMHA in vivo correlates with and may contribute to the greater susceptibility of female rats to hepatocarcinogenesis and of male rats to development of tumors in the nasal epithelium following oral exposure to NMHA.

Inhibition of N-nitrosodimethylamine metabolism in rats by ether anesthesia.

Short-term exposure to diethyl ether strongly inhibits the metabolism of N-nitrosodimethylamine (NDMA). Twenty-six 6-week-old male Fischer 344 rats were exposed to ether vapor until their righting reflex was lost (approximately 2 min). The animals were removed from the ether and NDMA was immediately administered by i.v. bolus injection at a dose of 300 microgram/kg via a cannula surgically inserted 20 h earlier. A second group of 28 rats received injections of NDMA in an identical manner but without ether exposure. In the unanesthetized animals blood levels of NDMA declined with a half-life of 11 min; by contrast essentially constant blood levels of NDMA were observed in ether-treated animals for 120 min after removal from the anesthetic. The apparent total systemic clearance for the 5-h experiment was reduced from 43 ml/min/kg without ether to 5 ml/min/kg with ether. Diethyl ether has been found previously to inhibit the metabolism of other drugs requiring oxidative metabolism but the suppression of clearance documented here appears to be unusually pronounced. It is recommended that ether's potential for altering metabolic rates be carefully considered when planning or interpreting animal experiments.

Use of 3,4-dichlorobenzenethiol as a trapping agent for alkylating intermediates during in vitro metabolism of nitrosamines.

Our studies using 3,4-dichlorobenzenethiol as a probe for methylating agent production during exposure of N-nitrosodimethylamine to rat liver S-9 preparations produced results different from those of an investigation reported in the literature. Methyl-3,4-dichlorophenyl thioether was detected, but the quantities found were not significantly different from the background levels of methylation product detected in the absence of nitrosamine. Only about 10% of the thioether isolated after incubating N-nitrosodi[14C]methylamine as substrate was radioactive. The results indicate that the majority of the methyl groups transferred to the sulfur nucleophile in our experiments came from components of the incubation mixture other than the nitrosamine. Some artifactual methylation was also associated with the analytical procedure. We conclude that 3,4-dichlorobenzenethiol should be used with caution in studies of alkylation during the in vitro metabolism of carcinogenic nitrosamines.

Deuterium isotope effect on denitrosation and demethylation of N-nitrosodimethylamine by rat liver microsomes.

In an attempt to elucidate the molecular basis for the decrease in rat liver carcinogenicity and DNA-alkylating ability that accompanies deuteration of N-nitrosodimethylamine (NDMA), NDMA and its fully deuterated analogue ([2H6]NDMA) were incubated with acetone-induced rat liver microsomes. Rates for the competing metabolic routes, denitrosation and demethylation, were determined from colorimetric data on nitrite and formaldehyde generation, respectively. The Vmax calculated for demethylation of NDMA was 7.9 nmol/min/mg, while that for denitrosation was 0.83 nmol/min/mg. Deuteration of NDMA did not significantly change the Vmax for either pathway, but it did increase the Km for demethylation from 0.06 to 0.3 mM. The Km for denitrosation was also increased from 0.06 to 0.3 mM on deuteration, as determined by incubating an equimolar mixture of amino-15N-labeled NDMA with [2H6]NDMA and measuring the methyl[15N]amine:[2H3]methylamine ratio by derivatization-gas chromatography-mass spectrometry. The fact that the Km values for denitrosation were so similar to those for demethylation suggested that the two pathways were catalyzed by the same enzyme. The isotope effects calculated from these data [VmaxH/VmaxD approximately 1 and (Vmax/Km)H/(Vmax/Km)D approximately 5] show that microsomal metabolism of NDMA is not significantly shifted from demethylation to denitrosation on deuteration of substrate and may indicate a low commitment to catalysis for the enzyme. The results are consistent with the view that the metabolism of NDMA is initiated by formation of an alpha-nitrosamino radical which either combines with a hydroxyl radical to form the alpha-hydroxynitrosamine as the initial product of the demethylation pathway or fragments to nitric oxide and N-methylformaldimine as the first products of denitrosation.

Carcinogenicity of deuterium-labeled 1,2-dimethylhydrazine in mice.

To study the effect of deuterium substitution on the carcinogenicity of 1,2-dimethylhydrazine (DMH) in mice, two comparisons were made between DMH and its fully methyl-deuterated analogue, [2H6]DMH. In a lifetime study with the CBA strain, groups of 19-30 animals of each sex were dosed s.c. weekly with 8 mg/kg of either DMH or [2H6]DMH for 8, 16, or 32 weeks. In the second study, female CF-1 mice were given DMH or [2H6]DMH in 10 weekly s.c. doses of 12 mg/kg each (13.2 mg/kg for [2H6]DMH) and examined for colon tumors 36 weeks after the first dose. Deuteration significantly decreased tumor incidence in the colon of males (P less than 0.01) and the anal tissue of both sexes (P less than 0.05) but increased that of hepatomas and lung tumors in males (P less than 0.01). Substrate deuteration did not significantly affect overall incidence of any other tumor type, however, including hemangioendotheliomas and kidney tumors in both sexes, as well as colon, uterine, ovarian, liver, and lung tumors in females. The results indicate that C--H bond breakage is kinetically important in the activation of DMH to its ultimately carcinogenic form in organs such as the male colon (relative risk in DMH-versus [2H6]DMH-treated animals approximately equal to 6), and that inhibition of this process by substrate deuteration allows a diversionary mechanism having a smaller isotope effect to become relatively more extensive. The qualitatively different effect in other organs (e.g., kidney, relative risk approximately equal to 1) supports recent suggestions that the net mechanism of activation can differ from one target tissue to another, possibly by striking a different balance between parallel metabolic pathways. The lack of a significant isotope effect on overall colon tumor incidence in females of either strain suggests that differences in relative importance among competing enzymes may also be responsible for sexual dimorphism in tumor induction by DMH.

In vivo radiation protection by nitric oxide modulation.

Drugs that affect blood flow have been shown to be whole body radiation protectors. Using NG-nitro-L-arginine, a specific inhibitor of nitric oxide synthase, and the NO-releasing agent (C2H5)2N[N(O)NO-]Na+ (DEA/NO), we have studied the ability of NO to modulate whole body radiation toxicity in C3H mice. NG-Nitro-L-arginine given to mice between 15 and 60 min prior to radiation afforded significant protection from whole body irradiation, e.g., the estimated whole body irradiation dose required to kill 50% of mice by 30 days after radiation (LD50/30) in mice treated with NG-nitro-L-arginine 60 min before irradiation was 1051 cGy compared with a whole body radiation LD50/30 of 822 cGy in control mice (P < 0.00001). Treatment of mice with DEA/NO prior to whole body irradiation also significantly reduced toxicity; the estimated whole body radiation LD50/30 was 1063 and 945 cGy in mice treated with DEA/NO 10 or 30 min before irradiation, respectively (P < 0.00001 for radiation LD50/30 of either DEA/NO-treated group compared with control). Measurement of [14C]etanidazole binding to bone marrow demonstrated that DEA/NO and NG-nitro-L-arginine exacerbated bone marrow hypoxia. Perturbations of NO levels have profound effects on in vivo radiosensitivity of normal tissues. We hypothesize that alterations in regional blood flow may underlie the changes in radiosensitivity that we have observed.

Metabolic denitrosation of N-nitrosodimethylamine in vivo in the rat.

Enzymatic denitrosation is a potentially inactivating metabolic route that has been shown to convert carcinogenic N-nitrosodimethylamine (NDMA) to methylamine (MA) in vitro. To investigate its quantitative course in vivo, groups of 8-week-old male Fischer rats have been given small (8-15 mumol/kg) p.o. or i.v. bolus doses of 14C-labeled NDMA and the subsequent formation of radioactive MA has been monitored by high performance liquid chromatographic analysis of serially collected blood samples from each individual. Adjusting the [14C]MA fluxes observed for the previously measured rates at which MA is itself eliminated from the system after intragastric administration, denitrosation was calculated to represent a rather uniform 21.3 +/- 1.3% (SE) of total NDMA elimination in the four animals studied. By contrast, repetition of the experiment with fully deuterated NDMA (NDMA-d6) revealed a significantly wider variance in the results (39.8 +/- 8.9%). An alternative calculation using values for elimination of i.v. doses of MA and its trideuteromethyl analogue gave an even larger difference for MA formation between NDMA and NDMA-d6, the estimated extents of in vivo denitrosation in this case being 14.5 +/- 0.9% and 48.3 +/- 10.8%, respectively. The results indicate that denitrosation is a major metabolic pathway for NDMA elimination and suggest that deuteration of the carcinogen induces a shift in its metabolism toward increasing denitrosation at the expense of the competing activation pathway. Consequently, denitrosation may be the previously undefined in vivo metabolic route, the existence of which was suggested by the findings that deuteration of NDMA lowered its hepatocarcinogenicity and liver DNA alkylating ability in rats.

Low-dose in vivo pharmacokinetic and deuterium isotope effect studies of N-nitrosodimethylamine in rats.

The rates of elimination of N-nitrosodimethylamine (NDMA) and its fully deuterated analogue (N-nitrosodi[2H6]methylamine, [2H6]NDMA) were studied in vivo to explore the origins of the difference in their carcinogenicity. Male Fischer 344 rats, 7.5 weeks of age, were given nitrosamine bolus doses of 1.35 mumol/kg by tail vein injection and 2.02 or 4.05 mumol/kg by p.o. gavage. Animals were sacrificed at various time points from 2.5 to 180 min after i.v. administration or 5 to 120 min after p.o. dosage, and their blood was analyzed for NDMA by gas chromatography-high resolution mass spectrometry. After i.v. injection, blood nitrosamine concentrations declined in an apparently biexponential manner with a terminal half-life of 10 min for NDMA and 12 min for [2H6]NDMA. The apparent total systemic blood clearances for NDMA and [2H6]NDMA were 39 and 26 ml/min/kg, respectively. The apparent steady-state volumes of distribution were nearly identical (297 and 309 ml/kg, respectively). The areas under the curve after 2.02- and 4.05-mumol/kg p.o. doses were proportional to dose. The apparent bioavailability of NDMA was 8%, while that of [2H6]NDMA was 21%. Isotope effects calculated as the ratios of first-pass metabolism, total systemic clearances, bioavailabilities, and intrinsic hepatic clearances were 1.2, 1.5, 2.6, and 3.2, respectively. The isotope effect determined from blood concentrations measured after simultaneous administration of NDMA and [2H6]NDMA by steady-state infusion (each at 1.5 mumol/kg/h) was 2.6 +/- 0.9 (SD). This study thus provides quantitative reference data on the time course of the disappearance of both N-nitrosodimethylamine and its deuterated analogue from blood (over 5 to 8 half-lives) after doses similar to those used to elicit liver tumors in chronic feeding studies, confirms the first-pass effect on their metabolism using direct blood measurements, and permits estimation of their bioavailabilities from actual blood concentrations. The results suggest that elimination pathways not involving alpha-hydroxylation are more important than is currently recognized.

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.