Integrated histopathological and urinary metabonomic investigation of the pathogenesis of microcystin-LR toxicosis.

Patterns of change of endogenous metabolites may closely reflect systemic and organ-specific toxic changes. The authors examined the metabolic effects of the cyanobacterial (blue-green algal) toxin microcystin-LR by (1)H-nuclear magnetic resonance (NMR) analysis of urinary endogenous metabolites. Rats were treated with a single sublethal dose, either 20 or 80 µg/kg intraperitoneally, and sacrificed at 2 or 7 days post dosing. Changes in the high-dose, 2-day sacrifice group included centrilobular hepatic necrosis and congestion, accompanied in some animals by regeneration and neovascularization. By 7 days, animals had recovered, the necrotizing process had ended, and the centrilobular areas had been replaced by regenerative, usually hypertrophic hepatocytes. There was considerable interanimal variation in the histologic process and severity, which correlated with the changes in patterns of endogenous metabolites in the urine, thus providing additional validation of the biomarker and biochemical changes. Similarity of the shape of the metabolic trajectories suggests that the mechanisms of toxic effects and recovery are similar among the individual animals, albeit that the magnitude and timing are different for the individual animals. Initial decreases in urinary citrate, 2-oxoglutarate, succinate, and hippurate concentrations were accompanied by a temporary increase in betaine and taurine, then creatine from 24 to 48 hours. Further changes were an increase in guanidinoacetate, dimethylglycine, urocanic acid, and bile acids. As a tool, urine can be repeatedly and noninvasively sampled and metabonomics utilized to study the onset and recovery after toxicity, thus identifying time points of maximal effect. This can help to employ histopathological examination in a guided and effective fashion.

Characterization of 2'-deoxyadenosine adducts derived from 4-oxo-2-nonenal, a novel product of lipid peroxidation.

Analysis of the reaction between 2'-deoxyadenosine and 4-oxo-2-nonenal by liquid chromatography/mass spectrometry revealed the presence of three major products (adducts A(1), A(2), and B). Adducts A(1) and A(2) were isomeric; they interconverted at room temperature, and they each readily dehydrated to form adduct B. The mass spectral characteristics of adduct B obtained by collision-induced dissociation coupled with multiple tandem mass spectrometry were consistent with those expected for a substituted etheno adduct. The structure of adduct B was shown by NMR spectroscopy to be consistent with the substituted etheno-2'-deoxyadenosine adduct 1' '-[3-(2'-deoxy-beta-D-erythropentafuranosyl)-3H-imidazo[2, 1-i]purin-7-yl]heptane-2' '-one. Unequivocal proof of structure came from the reaction of adducts A(1) and A(2) (precursors of adduct B) with sodium borohydride. Adducts A(1) and A(2) each formed the same reduction product, which contained eight additional hydrogen atoms. The mass spectral characteristics of this reduction product established that the exocyclic amino group (N(6)) of 2'-deoxyadenosine was attached to C-1 of the 4-oxo-2-nonenal. The reaction of 4-oxo-2-nonenal with calf thymus DNA was also shown to result in the formation of substituted ethano adducts A(1) and A(2) and substituted etheno adduct B. Adduct B was formed in amounts almost 2 orders of magnitude greater than those of adducts A(1) and A(2). This was in keeping with the observed stability of the adducts. The study presented here has provided additional evidence which shows that 4-oxo-2-nonenal reacts efficiently with DNA to form substituted etheno adducts.

Flavopiridol binds to duplex DNA.

Flavopiridol, the first potent cyclin-dependent kinase inhibitor to enter clinical trials, was recently found to be cytotoxic to noncycling cells. The present studies were performed to examine the hypothesis that flavopiridol, like several other antineoplastic agents that kill noncycling cells, might also interact with DNA. Consistent with this possibility, treatment of A549 human lung cancer cells with clinically achievable concentrations of flavopiridol resulted in rapid elevations of the DNA damage-responsive protein p53. In further studies, the binding of flavopiridol to DNA was examined in vitro by four independent techniques. Absorption spectroscopy revealed that addition of DNA to aqueous flavopiridol solutions resulted in a red shift of the flavopiridol lambda(max) from 311 to 344 nm, demonstrating an isosbestic point typical of changes seen with DNA-binding compounds. Reverse-phase high-performance liquid chromatography demonstrated that flavopiridol binds to genomic DNA to a similar extent as ethidium bromide and Hoechst 33258. Nuclear magnetic resonance spectroscopy revealed that DNA caused extreme broadening of flavopiridol 1H nuclear magnetic resonance signals that could be reversed by addition of ethidium bromide or by DNA melting, suggesting that flavopiridol binds to (and likely intercalates into) duplex DNA. Equilibrium dialysis demonstrated that the equilibrium dissociation constant of the flavopiridol-DNA complex (5.4+/-3.4 x 10(-4) M) was in the same range observed for binding of the intercalators doxorubicin and pyrazoloacridine to DNA. Molecular modeling confirmed the feasibility of flavopiridol intercalation into DNA and analysis of the effects of flavopiridol in the National Cancer Institute tumor cell line panel using the COMPARE algorithm demonstrated that flavopiridol most closely resembles cytotoxic antineoplastic intercalators. Collectively, these data suggest that DNA might be a second target of flavopiridol, providing a potential explanation for the ability of this agent to kill noncycling cancer cells.

Metabolism and excretion of [(14)C]celecoxib in healthy male volunteers.

We determined the disposition of a single 300-mg dose of [(14)C]celecoxib in eight healthy male subjects. The [(14)C]celecoxib was administered as a fine suspension reconstituted in 80 ml of an apple juice/Tween 80/ethanol mixture. Blood and saliva samples were collected at selected time intervals after dosing. All urine and feces were collected on the 10 consecutive days after dose administration. Radioactivity in each sample was determined by liquid scintillation counting or complete oxidation and liquid scintillation counting. Metabolic profiles in plasma, urine, and feces were obtained by HPLC, and metabolites were identified by mass spectrometry and NMR. [(14)C]Celecoxib was well absorbed, reaching peak plasma concentrations within 2 h of dosing. [(14)C]Celecoxib was extensively metabolized, with only 2.56% of the radioactive dose excreted as celecoxib in either urine or feces. The total percentage of administered radioactive dose recovered was 84.8 +/- 4.9%, with 27.1 +/- 2.2% in the urine and 57.6 +/- 7.3% in the feces. The oxidative metabolism of celecoxib involved hydroxylation of celecoxib at the methyl moiety followed by further oxidation of the hydroxyl group to form a carboxylic acid metabolite. The carboxylic acid metabolite of celecoxib was conjugated with glucuronide to form the 1-O-glucuronide. The percentages of the dose excreted in the feces as celecoxib and the carboxylic acid metabolite were 2.56 +/- 1.09 and 54.4 +/- 6.8%, respectively. The majority of the dose excreted in the urine was the carboxylic acid metabolite (18.8 +/- 2.1%); only a small amount was excreted as the acyl glucuronide (1.48 +/- 0.15%).

Isolation and identification of metabolites of leukotriene A4 hydrolase inhibitor SC-57461 in rats.

The metabolic fate of SC-57461, N-methyl-N-[3-[4-(phenylmethyl)-phenoxy]propyl]-beta-alanine, a potent and specific inhibitor of the leukotriene A4 hydrolase, was determined by LC/MS/MS, NMR and GC/MS in male Sprague-Dawley rats. The major metabolites of SC-57461 in rats were the desmethyl metabolite, the hydroxylated metabolite, the N-oxide metabolite, the hydroxylamine metabolite, and the propionic acid metabolite. The N-oxide metabolite was found to be stable in the rat plasma and urine, but was unstable in most organic solvents (methanol, acetonitrile, and methylene chloride, etc.) because of the classic Cope reaction of the N-oxide, which led to the formation of the corresponding hydroxylamine product and acrylic acid. The hydroxylamine metabolite and acrylic acid were reactive in the biomatrix and could not be isolated in the in vivo samples. However, formation of the hydroxylamine metabolite and acrylic acid from the N-oxide metabolite in methylene chloride was verified by NMR. The propionic acid metabolite was found to be the common metabolite shared by SC-57461, N-oxide metabolite, as well as the hydroxylamine metabolite, which suggested a sequential metabolism of SC-57461 in rats. The ultimate fate of the propionic acid metabolite was incorporation into rat glycerolipid metabolism as a result of its structural similarity to aryl-substituted propionic acid, a known class of compounds that can be incorporated into rat glycerolipid metabolism. Finally, the isolated hydroxylated metabolite and the N-desmethyl metabolite were found to have excellent inhibitory effects toward leukotriene A4 hydrolase and therefore were the major active metabolites of SC-57461 in rats.

Difference in metabolic profile of potassium canrenoate and spironolactone in the rat: mutagenic metabolites unique to potassium canrenoate.

The metabolic fates of potassium canrenoate (PC) and spironolactone (SP) were compared for the rat in vivo and in vitro. Approximately 18% of an in vivo dose of SP was metabolized to canrenone (CAN) and related compounds in the rat. In vitro, 20-30% of SP was dethioacetylated to CAN and its metabolites by rat liver 9000 g supernatant (S9). Thus, the major route of SP metabolism is via pathways that retain the sulfur moiety in the molecule. PC was metabolized by rat hepatic S9 to 6 alpha, 7 alpha- and 6 beta, 7 beta-epoxy-CAN. The beta-epoxide was further metabolized to its 3 alpha- and 3 beta-hydroxy derivatives as well as its glutathione (GSH) conjugate. Both 3 alpha- and 3 beta-hydroxy-6 beta, 7 beta-epoxy-CAN were shown to be direct acting mutagens in the mouse lymphoma assay, whereas 6 alpha, 7 alpha- and 6 beta, 7 beta-epoxy-CAN were not. These mutagenic metabolites, their precursor epoxides and their GSH conjugates were not formed from SP under identical conditions. The above findings appear to be due to inhibition of metabolism of CAN formed from SP by SP and/or its S-containing metabolites, since the in vitro metabolism of PC by rat hepatic microsomes was appreciably reduced in the presence of SP. The hypothesized mechanism(s) for this inhibition is that SP and its S-containing metabolites specifically inhibit an isozyme of hepatic cytochrome P-450 or SP is a preferred substrate over PC/CAN for the metabolizing enzymes. Absence of the CAN epoxide pathway in the metabolism of SP provides a possible explanation for the observed differences in the toxicological profiles of the two compounds.

The pH dependence of disobutamide-induced clear cytoplasmic vacuoles in cultured cells.

Cellular uptake of disobutamide (D), and clear cytoplasmic vacuoles (CCV) induction by D in cultured rat urinary bladder carcinoma cells were dependent on the culture medium pH. At pH 6.0-6.7, drug uptake was slow and no CCV formed in 24 hr. At pH 7.0-8.0, the rate of D uptake and early appearance of CCV were directly proportional to increased basicity. This was explained by the increasing fraction of un-ionized D molecules at increasing basicity of the culture medium. It is only these electrically neutral D molecules which can penetrate the lipoidal cell membrane to induce formation of CCV. Intracellular presence of D was demonstrated by mass spectrometry methods. The results indicate that D is incorporated intracellularly, that D and not its metabolite(s) is in cells, and suggest that CCV are a result of drug sequesteration.

The analysis and source of 1-diphenylmethyl-4-nitrosopiperazine in 1-diphenylmethyl-4-[(6-methyl-2-pyridyl) methyleneamino]piperazine: a case history.

Analytical methods were developed to determine whether there was less than one ppm of II in IV. Purified IV contained a compound with the same GC retention time as authentic II on OV-17 and Silar 10C columns, using a FID or a nitrogen/phosphorus detector. The GC-coupled mass spectra contained major peaks at m/e 251 and 167 and the GC-coupled methane CI spectra gave the quasi-molecular ion, m/e 282. However, the CI selected ion monitor indicated three compounds at m/e 282. The nitrosamine II was separated from IV by HPLC on muBondapak C18. The GC-coupled mass spectra of the HPLC nitrosamine fraction had peaks at m/e 251 and 167 and other peaks for a compound of greater MW than II, although the GC retention times were the same. Evidence for II and a decrease in the detection limit to one ppm was obtained with a TEA interfaced with a HPLC. To determine if II was formed from IV, it was exposed to ozone at -78 degrees C in methylene chloride. The DSC, IR and NMR of the product were coincident with those of the standard of II. In other experiments, IV in methylene chloride was exposed to light and dry air in the presence and absence of methylene blue. The PMR and 13C NMR of the product formed in the presence of methylene blue were the same as that of II. It is postulated that this nitrosamine was formed by a singlet oxygen mechanism.

An animal model for screening drugs for antipsoriatic properties using hydroxyapatite to isolate DNA rapidly from the epidermis.

A new, rapid, simple and accurate procedure for extracting DNA from the epidermis is described involving hydroxyapatite. This compound has an unique property of high affinity for double stranded native DNA, but none for RNA and protein. The technique has been applied to a hairless mouse model for screening systemic and topical agents for cell inhibitory properties in proliferating epidermis for potential use in the therapy of psoriasis. Mitotic counts, autoradiography and a standard chloroform procedure for isolating DNA have confirmed the results obtained using hydroxyapatite column chromatography. In addition, compounds which are known to be effective in psoriasis, have been found to be effective in the system suggesting that it should be a good predictive screen for new therapies. Of several compounds screened for topical activity, cycloheximide has been found to be very effective in the system and preliminary clinical studies confirm its efficacy in psoriasis.