Age-related differences in susceptibility to cisplatin-induced renal toxicity.

Limited experimental models exist to assess drug toxicity in pediatric populations. We recently reported how a multi-age rat model could be used for pre-clinical studies of comparative drug toxicity in pediatric populations. The objective of this study was to expand the utility of this animal model, which previously demonstrated an age-dependent sensitivity to the classic nephrotoxic compound, gentamicin, to another nephrotoxicant, namely cisplatin (Cis). Sprague-Dawley rats (10, 25, 40 and 80 days old) were injected with a single dose of Cis (0, 1, 3 or 6 mg kg(-1) i.p.). Urine samples were collected prior and up to 72 h after treatment in animals that were >or= 25 days old. Several serum, urinary and 'omic' injury biomarkers as well as renal histopathology lesions were evaluated. Statistically significant changes were noted with different injury biomarkers in different age groups. The order of age-related Cis-induced nephrotoxicity was different than our previous study with gentamicin: 80 > 40 > 10 > 25 day-old vs 10 >or= 80 > 40 > 25-day-old rats, respectively. The increased levels of kidney injury molecule-1 (Kim-1: urinary protein/tissue mRNA) provided evidence of early Cis-induced nephrotoxicity in the most sensitive age group (80 days old). Levels of Kim-1 tissue mRNA and urinary protein were significantly correlated to each other and to the severity of renal histopathology lesions. These data indicate that the multi-age rat model can be used to demonstrate different age-related sensitivities to renal injury using mechanistically distinct nephrotoxicants, which is reflected in measurements of a variety of metabolite, gene transcript and protein biomarkers.

Identification of C-glycoside flavonoids as potential mutagenic compounds in kava.

Kava (Piper methysticum) extract products have been implicated in a number of severe hepatotoxicity cases. However, systematic toxicological studies regarding kava consumption have not been reported. In this study, 6 major kavalactones and different solvent fractions of kava roots, leaves, and stem peelings were evaluated for their mutagenic potential. None of the kavalactones was found to be positive in the experimental concentration ranges tested by the umu test (a sensitive test for point mutations). However, among the different solvent fractions, the n-butanol fraction of kava leaves was positive. Further investigations using bioassay-directed isolation and analysis indicated that 2 C-glycoside flavonoid compounds accounted for the positive mutagenic results. Two isolated compounds were identified as 2''-O-rhamnosylvitexin and schaftoside by NMR and MS techniques.

Metabolomic study of cisplatin-induced nephrotoxicity.

We have shown that cisplatin inhibits fatty acid oxidation, and that fibrate treatment ameliorates renal function by preventing the inhibition of fatty acid oxidation and proximal tubule cell death. Urine samples of mice treated with single injection of cisplatin (20 mg/kg body weight) were collected for 3 days and analyzed by 1H-nuclear magnetic resonance (NMR) spectroscopy. In a separate group, urine samples of mice treated with peroxisome proliferator-activated receptor-alpha (PPARalpha) ligand WY were also analyzed by NMR after 2 days of cisplatin exposure. Biochemical analysis of endogenous metabolites was performed in serum, urine, and kidney tissue. Electron microscopic studies were carried out to examine the effects of PPARalpha ligand and cisplatin. Principal component analysis demonstrated the presence of glucose, amino acids, and trichloacetic acid cycle metabolites in the urine after 48 h of cisplatin administration. These metabolic alterations precede changes in serum creatinine. Biochemical studies confirmed the presence of glucosuria, but also demonstrated the accumulation of nonesterified fatty acids, and triglycerides in serum, urine, and kidney tissue, in spite of increased levels of plasma insulin. These metabolic alterations were ameliorated by the use of PPARalpha ligand. Electron microscopic analysis confirmed the protective effect of the fibrate on preventing cisplatin-mediated necrosis of the S3 segment of the proximal tubule. Our study shows that cisplatin-induces a unique NMR metabolic profile in urine of mice that developed acute renal failure, and confirms the protective effect of a fibrate class of PPARalpha ligands. We propose that the injury-induced metabolic profile may be used as a biomarker of cisplatin-induced nephrotoxicity.

Developing 13C NMR quantitative spectrometric data-activity relationship (QSDAR) models of steroid binding to the corticosteroid binding globulin.

We have developed four quantitative spectrometric data-activity relationship (QSDAR) models for 30 steroids binding to corticosteroid binding globulin, based on comparative spectral analysis (CoSA) of simulated 13C nuclear magnetic resonance (NMR) data. A QSDAR model based on 3 spectral bins had an explained variance (r2) of 0.80 and a cross-validated variance (q2) of 0.78. Another QSDAR model using the 3 atoms from the comparative structurally assigned spectral analysis (CoSASA) of simulated 13C NMR on a steroid backbone template gave an explained variance (r2) of 0.80 and a cross-validated variance (q2) of 0.73. Positions 3 and 14 from the steroid backbone template have correlations with the relative binding activity to corticosteroid binding globulin that are greater than 0.52. The explained correlation and cross-validated correlation of these QSDAR models are as good as previously published quantitative structure-activity relationship (QSAR), self-organizing map (SOM) and electrotopological state (E-state) models. One reason that the cross-validated variance of QSDAR models were as good as the other models is that simulated 13C NMR spectral data are more accurate than the errors introduced by the assumptions and approximations used in calculated electrostatic potentials, E-states, HE-states, and the molecular alignment process of QSAR modeling. The QSDAR models developed provide a rapid, simple way to predict the binding activity of a steroid to corticosteroid binding globulin.

Models of polychlorinated dibenzodioxins, dibenzofurans, and biphenyls binding affinity to the aryl hydrocarbon receptor developed using (13)c NMR data.

Quantitative spectroscopic data-activity relationship (QSDAR) models for polychlorinated dibenzofurans (PCDFs), dibenzodioxins (PCDDs), and biphenyls (PCBs) binding to the aryl hydrocarbon receptor (AhR) have been developed based on simulated (13)C nuclear magnetic resonance (NMR) data. All the models were based on multiple linear regression of comparative spectral analysis (CoSA) between compounds. A 1.0 ppm resolution CoSA model for 26 PCDF compounds based on chemical shifts in five bins had an explained variance (r(2)) of 0.93 and a leave-one-out (LOO) cross-validated variance (q(2)) of 0.90. A 2.0 ppm resolution CoSA model for 14 PCDD compounds based on chemical shifts in five bins had an r(2) of 0.91 and a q(2) of 0.81. The 1.0 ppm resolution CoSA model for 12 PCB compounds based on chemical shifts in five bins had an r(2) of 0.87 and a q(2) of 0.45. The models with more compounds had a better q(2) because there are more multiple chemical shift populated bins available on which to base the linear regression. A 1.0 ppm resolution CoSA model for all 52 compounds that was based on chemical shifts in 12 bins had an r(2) of 0.85 and q(2) of 0.71. A canonical variance analysis of the 1.0 ppm CoSA model for all 52 compounds when they were separated into 27 strong binding and 25 weak binding compounds was 98% correct. Conventional quantitative structure-activity relationship (QSAR) modeling suffer from errors introduced by the assumptions and approximations involved in calculated electrostatic potentials and the molecular alignment process. QSDAR modeling is not limited by such errors since electrostatic potential calculations and molecular alignment are not done. The QSDAR models provide a rapid, simple and valid way to model the PCDF, PCDD, and PCB binding activity in relation to the aryl hydrocarbon receptor (AhR).

(13)C NMR quantitative spectrometric data-activity relationship (QSDAR) models of steroids binding the aromatase enzyme.

Five quantitative spectroscopic data-activity relationships (QSDAR) models for 50 steroidal inhibitors binding to aromatase enzyme have been developed based on simulated (13)C nuclear magnetic resonance (NMR) data. Three of the models were based on comparative spectral analysis (CoSA), and the two other models were based on comparative structurally assigned spectral analysis (CoSASA). A CoSA QSDAR model based on five principal components had an explained variance (r(2)) of 0.78 and a leave-one-out (LOO) cross-validated variance (q(2)) of 0.71. A CoSASA model that used the assigned (13)C NMR chemical shifts from a steroidal backbone at five selected positions gave an r(2) of 0.75 and a q(2) of 0.66. The (13)C NMR chemical shifts from atoms in the steroid template position 9, 6, 3, and 7 each had correlations greater than 0.6 with the relative binding activity to the aromatase enzyme. All five QSDAR models had explained and cross-validated variances that were better than the explained and cross-validated variances from a five structural parameter quantitative structure-activity relationship (QSAR) model of the same compounds. QSAR modeling suffers from errors introduced by the assumptions and approximations used in partial charges, dielectric constants, and the molecular alignment process of one structural conformation. One postulated reason that the variances of QSDAR models are better than the QSAR models is that (13)C NMR spectral data, based on quantum mechanical principles, are more reflective of binding than the QSAR model's calculated electrostatic potentials and molecular alignment process. The QSDAR models provide a rapid, simple way to model the steroid inhibitor activity in relation to the aromatase enzyme.

Metabolism of the veterinary fluoroquinolone sarafloxacin by the fungus Mucor ramannianus.

To investigate the microbial biotransformation of veterinary fluoroquinolones, Mucor ramannianus was grown in sucrose/peptone broth with sarafloxacin for 18 days. Cultures were extracted with ethyl acetate and extracts were analyzed by liquid chromatography. The two metabolites (26% and 15% of the A280, respectively) were identified by mass and 1H nuclear magnetic resonance spectra as N-acetylsarafloxacin and desethylene-N-acetylsarafloxacin. The biological formation of desethylene-N-acetylsarafloxacin has not been previously observed.

Use of 13C NMR spectrometric data to produce a predictive model of estrogen receptor binding activity.

We have developed a spectroscopic data-activity relationship (SDAR) model based on 13C NMR spectral data for 30 estrogenic chemicals whose relative binding affinities (RBA) are available for the alpha (ERalpha) and beta (ERbeta) estrogen receptors. The SDAR models segregated the 30 compounds into strong and medium binding affinities. The SDAR model gave a leave-one-out (LOO) cross-validation of 90%. Two compounds that were classified incorrectly in the SDAR model were in the transition zone between classifications. Real and predicted 13C NMR chemical shifts were used with test compounds to evaluate the predictive behavior of the SDAR model. The 13C NMR SDAR model using predicted 13C NMR data for the test compounds provides a rapid, reliable, and simple way to screen whether a compound binds to the estrogen receptors.

Development of a (32)P-postlabeling/HPLC method for detection of dehydroretronecine-derived DNA adducts in vivo and in vitro.

Pyrrolizidine alkaloids are naturally occurring genotoxic chemicals produced by a large number of plants. Metabolism of pyrrolizidine alkaloids in vivo and in vitro generates dehydroretronecine (DHR) as a common reactive metabolite. In this study, we report the development of a (32)P-postlabeling/HPLC method for detection of (i) two DHR-3'-dGMP and four DHR-3'-dAMP adducts and (ii) a set of eight DHR-derived DNA adducts in vitro and in vivo. The approach involves (1) synthesis of DHR-3'-dGMP, DHR-3'-dAMP, and DHR-3',5'-dG-bisphosphate standards and characterization of their structures by mass and (1)H NMR spectral analyses, (2) development of optimal conditions for enzymatic DNA digestion, adduct enrichment, and (32)P-postlabeling, and (3) development of optimal HPLC conditions. Using this methodology, we have detected eight DHR-derived DNA adducts, including the two epimeric DHR-3',5'-dG-bisphosphate adducts both in vitro and in vivo.

Producing 13C NMR, infrared absorption, and electron ionization mass spectrometric data models of the monodechlorination of chlorobenzenes, chlorophenols, and chloroanilines.

We have developed four spectroscopic data-activity relationship (SDAR) models of monodechlorination of 32 chlorinated benzene compounds in anaerobic estuarine sediment. The SDAR models were based on combinations of 13C nuclear magnetic resonance (NMR), infrared absorption (IR), and electron ionization mass spectrometric (EI MS) data. The SDAR models segregated the 32 compounds into 17 readily monodechlorinated compounds and 15 not readily monodechlorinated compounds. The SDAR model based on 13C NMR, IR, and EI MS data gave a leave-one-out cross-validation of 93.8%. The SDAR model based on a composite of 13C NMR and IR data gave a leave-one-out cross-validation of 90.6%. The SDAR model based on a composite of IR and EI MS data gave a leave-one-out cross-validation of 84.4%. The SDAR model based on a composite of 13C NMR and EI MS data gave a leave-one-out cross-validation of 84.4%. These reliable SDAR models provide a rapid and simple way to predict whether a chlorinated benzene compound will readily go through monodechlorination. The FDA has filed a patent application on methods of using any combination of spectral data (NMR, MS, UV-vis, IR, and fluorescence, phosphorescence) to model a chemical, physical, or biological endpoint.

(13)C NMR and electron ionization mass spectrometric data-activity relationship model of estrogen receptor binding.

Two Spectroscopic Data-Activity Relationship (SDAR) models based on (13)C nuclear magnetic resonance (NMR) and electron ionization mass spectra (EI MS) data were developed for 108 compounds whose relative binding affinities (RBA) to the estrogen receptor are known. The (13)C NMR and EI MS data were used as spectrometric digital fingerprints to reflect the electronic and structural characteristics of the compounds. Both SDAR models segregated the 108 compounds into 20 strong, 15 medium, and 73 weak relative binding classifications. The first SDAR model, based on (13)C NMR data alone, gave a leave-one-out (LOO) cross-validation of 75.0%. The second SDAR model, based on a composite of (13)C NMR and EI MS data, gave a LOO cross-validation of 82.4%. Many of the misidentifications from the cross-validations were between medium and weak classifications, where there were fewer specific spectrometric characteristics to identify the relationship of spectra to estrogen receptor binding. Real and predicted (13)C NMR chemical shifts were used to test the predictive behavior of both SDAR models. The ease of use and speed of SDAR modeling may facilitate their use with other toxicological endpoints.

DNA adduct formation by Fusarium culture extracts: lack of role of fusarin C.

Fusarium fungi have been shown to infect corn and other crops worldwide, and have a significant impact on human health through loss of crops or contamination of food with mycotoxins. Isolates of Fusarium fungi from an area of South Africa with high incidence of esophageal cancer have been shown to induce esophageal and liver cancer in rats. Several isolates of Fusarium fungi were grown on corn to determine if genotoxic products were produced. We report the incubation of methanol extracts of Fusarium verticillioides cultures with DNA in the presence of rat liver fractions (S9) resulted in the formation of a unique DNA adduct that was detected by (32)P-postlabeling. Fusarin C was purified from cultures of Fusarium verticillioides RRC 415, and was not responsible for the formation of the DNA adduct. Treatment of the methanolic extracts with ultraviolet B radiation reduced the fusarin C content in the extract; however, this had no effect on the formation of the DNA adduct following incubation of the extract with DNA and S9. The unique DNA adduct was formed following the incubation of several Fusarium verticillioides isolates from the US and South Africa, while extracts of cultures of Fusarium graminearium and Fusarium sacchari isolates formed very little of the DNA adduct when incubated with DNA and S9. These data suggest that neither fusarin C nor any of its metabolites are responsible for formation of the DNA adduct, and that an unidentified compound is present in F. verticillioides cultures that forms a DNA adduct, and may be important in the etiology of human esophageal cancer.

Evaluation of major active components in St. John's Wort dietary supplements by high-performance liquid chromatography with photodiode array detection and electrospray mass spectrometric confirmation.

A RP-HPLC method with photodiode array detection and LC-electrospray ionization (ESI) MS confirmation was established for the determination of major active components in St. John's Wort dietary supplement capsules. The samples alternatively were extracted with ethanol-acetone (2:3) using a 55 degrees C water-bath shaker or an ambient temperature ultrasonic bath. Extracts were separated by RP-C18 chromatography using a 95-min water-methanol-acetonitrile-trifluoroacetic acid gradient. The major components were identified by photodiode array detection and then confirmed by LC-ESI-MS. The quantification of components was performed using an internal standard (luteolin). This method may serve as a valuable tool for the quality evaluation of St. John's Wort dietary supplement products.

Microbiological transformation of enrofloxacin by the fungus Mucor ramannianus.

Enrofloxacin metabolism by Mucor ramannianus was investigated as a model for the biotransformation of veterinary fluoroquinolones. Cultures grown in sucrose-peptone broth were dosed with enrofloxacin. After 21 days, 22% of the enrofloxacin remained. Three metabolites were identified: enrofloxacin N-oxide (62% of the total absorbance), N-acetylciprofloxacin (8.0%), and desethylene-enrofloxacin (3.5%).

Isolation of human intestinal bacteria metabolizing the natural isoflavone glycosides daidzin and genistin.

Fecal bacteria from a healthy individual were screened for the specific bacteria involved in the metabolism of dietary isoflavonoids. Two strains of bacteria capable of producing primary and secondary metabolites from the natural isoflavone glycosides daidzin and genistin were detected. The metabolites were identified by comparison of their HPLC/mass, 1H NMR and UV spectra with those of standard and synthetic compounds. Both Escherichia coli HGH21 and the gram-positive strain HGH6 converted daidzin and genistin to the their respective aglycones daidzein and genistein. Under anoxic conditions, strain HGH6 further metabolized the isoflavones daidzein and genistein to dihydrodaidzein and dihydrogenistein, respectively. The reduction of a double bond between C-2 and C-3 to a single bond was isoflavonoid-specific by strain HGH6, which did not reduce a similar bond in the flavonoids apigenin and chrysin. Strain HGH6 did not further metabolize dihydrodaidzein and dihydrogenistein. This is the first study in which specific colonic bacteria that are involved in the metabolism of daidzin and genistin have been detected.

Biotransformation of vinclozolin by the fungus Cunninghamella elegans.

This study investigated the biotransformation of the dicarboximide fungicide vinclozolin [3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione] by the fungus Cunninghamella elegans. Experiments with phenyl-[U-ring-14C]vinclozolin showed that after 96 h incubation, 93% had been transformed to four major metabolites. Metabolites were separated by HPLC and characterized by mass and NMR spectroscopy. Biotransformation occurred predominantly on the oxazolidine-2,4-dione portion of vinclozolin. The metabolites were identified as the 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide, N-(2-hydroxy-2-methyl-1-oxobuten-3-yl)-3,5-dichlorophenyl-1-carbamic acid, and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide. The enanilide compound has been reported previously as a plant and mammalian metabolite and is implicated to contain antiandrogenic activity. The 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide are novel metabolites.

Regioselective transformation of ciprofloxacin to N-acetylciprofloxacin by the fungus Mucor ramannianus.

A strain of the saprobic fungus Mucor ramannianus, isolated from a forest, was used to demonstrate the potential for ciprofloxacin biotransformation by zygomycetes in the environment. The fungus carried out the regioselective N-acetylation of ciprofloxacin to a single product, which was purified from culture extracts by high-performance liquid chromatography. The metabolite was identified by mass and nuclear magnetic resonance spectrometry as 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-(4-acetyl-1-piperazinyl)-3- quinolinecarboxylic acid.

Determination of internuclear angles of DNA using paramagnetic-assisted magnetic alignment.

Paramagnetic ions have been used to assist the magnetic alignment of DNA. The anisotropy of the binding sites is sufficient to give rise to significant alignment of the DNA with the observed proton-carbon dipolar couplings spanning a 70-Hz range. The dipolar couplings have been used to determine the positions of the axial and rhombic alignment axes. The positions of the alignment axes relative to the positions of the binding sites of the paramagnetic europium ions have also been determined.

Interresidue quiet NOEs for DNA structural studies.

The potential utility of long-range NOEs in DNA has not been exploited since the observed signals have contributions both from the direct magnetization route and from multiple diffusion pathways. The Quiet NOE approach can be used to select for the direct magnetization transfer pathway by suppressing spin diffusion. A single-band Quiet NOE, which allows detection of the direct NOEs between protons in a selected chemical shift window, has been demonstrated on two duplex DNAs, and the NOEs observed can contain important structural information.

Structures of apurinic and apyrimidinic sites in duplex DNAs.

Natural and exogenous processes can give rise to abasic sites with either a purine or pyrimidine as the base on the opposing strand. The solution state structures of the apyrimidinic DNA duplex, with D6 indicating an abasic site, [sequence: see text] referred to as AD, and the apurinic DNA duplex with a dC17, referred to as CD, have been determined. A particularly striking difference is that the abasic site in CD is predominantly a beta hemiacetal, whereas in AD the alpha and beta forms are equally present. Hydrogen bonding with water by the abasic site and the base on the opposite strand appears to play a large role in determining the structure near the damaged site. Comparison of these structures with that of a duplex DNA containing a thymine glycol at the same position as the abasic site and with that of a duplex DNA containing an abasic site in the middle of a curved DNA sequence offers some insight into the common and distinct structural features of damaged DNA sites.