Toxicogenomics and cancer risk assessment: a framework for key event analysis and dose-response assessment for nongenotoxic carcinogens.

In order to determine a threshold for nongenotoxic carcinogens, the traditional risk assessment approach has been to identify a mode of action (MOA) with a nonlinear dose-response. The dose-response for one or more key event(s) linked to the MOA for carcinogenicity allows a point of departure (POD) to be selected from the most sensitive effect dose or no-effect dose. However, this can be challenging because multiple MOAs and key events may exist for carcinogenicity and oftentimes extensive research is required to elucidate the MOA. In the present study, a microarray analysis was conducted to determine if a POD could be identified following short-term oral rat exposure with two nongenotoxic rodent carcinogens, fenofibrate and methapyrilene, using a benchmark dose analysis of genes aggregated in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) biological processes, which likely encompass key event(s) for carcinogenicity. The gene expression response for fenofibrate given to rats for 2days was consistent with its MOA and known key events linked to PPARα activation. The temporal response from daily dosing with methapyrilene demonstrated biological complexity with waves of pathways/biological processes occurring over 1, 3, and 7days; nonetheless, the benchmark dose values were consistent over time. When comparing the dose-response of toxicogenomic data to tumorigenesis or precursor events, the toxicogenomics POD was slightly below any effect level. Our results suggest that toxicogenomic analysis using short-term studies can be used to identify a threshold for nongenotoxic carcinogens based on evaluation of potential key event(s) which then can be used within a risk assessment framework.

Direct analysis of microbial extracts containing metabolites of ethylenediamine-type antihistamines via high-performance liquid chromatography-thermospray mass spectrometry.

The utility of high-performance liquid chromatography-thermospray mass spectrometry (HPLC-TSMS) for the characterization of the ethylenediamine-type antihistamines, pyrilamine, methapyrilene, tripelennamine, and thenyldiamine, and their methylene chloride-extractable microbial metabolites from a biological matrix is demonstrated. Typically, the [M + H]+ ion was detected as the base peak in the TS mass spectra of these compounds. The ethylenediamine-type antihistamine metabolites were detected in an extract of a fungal culture grown in the presence of 5 mg of the antihistamine. A detection limit of 200 ng was observed for the HPLC-TSMS analysis of pyrilamine.

Fast-atom bombardment and thermospray mass spectrometry for the characterization of two glucuronide metabolites of methapyrilene.

Two conjugated metabolites of methapyrilene hydrochloride isolated from mouse-hepatocytes were examined by mass spectrometry using fast-atom bombardment (FAB) and thermospray ionization. The major metabolite, methapyrilene glucuronide, was identified based on a prominent peak due to the protonated molecule as well as the loss of the dimethylamine and sugar moieties. Identification of the second metabolite was complicated by large signals associated with the biological sample matrix. The complementary nature of the fragmentation observed in the mass spectra using FAB and thermospray ionization allowed this metabolite to be identified as the desmethylmethapyrilene glucuronide. The fragmentation observed using FAB ionization was not greatly affected by the presence of the glucuronide moiety. While loss of the sugar moiety indicated a glucuronide, additional fragmentation confirmed the presence of the underlying ethylenediamine substructure which is characteristic of this class of antihistamines.

Characterization of the antihistamines tripelennamine, methapyrilene, and thenyldiamine and their N-oxides by thermospray mass spectrometry.

This study describes an investigation of the thermospray (TS) mass spectrometric analysis of tripelennamine, methapyrilene, thenyldiamine, and their N-oxide derivatives. These compounds were analyzed by direct injection TS mass spectrometry in the column bypass mode and with 0.1M ammonium acetate/methanol (80:20, v/v) as the mobile phase. Typically, the parent antihistamines produced only [MH]s ions under these conditions. The N-oxides provided strong [MH]s ions and multiple fragment ions. A scheme to explain the fragmentation patterns is proposed.

Metabolism of methapyrilene by rat-liver homogenate.

The metabolism of methapyrilene(I) in rat-liver 9000 g supernatant fraction produced four new metabolites positively identified by comparison of g.l.c. retention times and mass-spectral fragmentation patterns with those of authentic materials. These compounds are 2-thiophene-methanol(VI), 2-thiophenecarboxylic acid(VII), N-2-pyridyl-N'-dimethylethylenediamine(IX) and 2-aminopyridine(X). In addition, the previously known metabolite 2-[(2-thienylmethyl)amino]-pyridine(VIII) was also positively identified. Six other metabolites were tentatively identified by analysis of the mass-spectral fragmentation patterns of both the trimethylsilyl and the tertiary butyldimethylsilyl derivatives of each compound. These compounds are tentatively identified as: normethapyrilene(II), (hydroxypyridyl)-methapyrilene(XII), methapyrilenamide(XIV), (hydroxypyridyl)-normethapyrilene(XVI), (hydroxypyridyl)-desmethylmethapyrilenamide(XVII), and (hydroxypyridyl) methapyrilenamide(XVIII). Quantification of II, VI-X, XII and XIV account for approx. 65% of the metabolized methapyrilene.

Stability of dobutamine hydrochloride and verapamil hydrochloride in 0.9% sodium chloride and 5% dextrose injections.

The stability of dobutamine hydrochloride (250 micrograms/ml) and verapamil hydrochloride (160 micrograms/ml) alone and in combination in 0.9% sodium chloride injection or 5% dextrose injection was studied. Solutions were stored both in plastic i.v. bags and in amber-colored glass bottles at 24 degrees C and 5 degrees C for up to seven days. Before storage and at various times during storage, solutions were assayed at least in triplicate by high-performance liquid chromatography, pH was recorded, and visual appearance was noted. All solutions tested under all conditions retained at least 90% potency for seven days. In plastic i.v. bags, dobutamine either alone or in combination with verapamil in both diluents turned a light-pink color in 24 hours at 24 degrees C. The intensity of the pink color increased with time in 0.9% sodium chloride injection; in 5% dextrose injection, solutions, became clear in 48 hours. The pH of solutions prepared in plastic i.v. bags in 5% dextrose injection decreased from 4.0 to 3.1 during the seven-day period at 24 degrees C; results for solutions in amber bottles were similar. At 5 degrees C, the pH and clarity of all solutions in bags and bottles remained stable for seven days. At the concentrations tested, dobutamine hydrochloride combined with verapamil hydrochloride is stable in 0.9% sodium chloride injection and 5% dextrose injection for 48 hours at 24 degrees C and for seven days at 5 degrees C.

Trace analysis of the antihistamines methapyrilene hydrochloride, pyrilamine maleate and triprolidine hydrochloride monohydrate in animal feed, human urine and wastewater by high-performance liquid chromatography and gas chromatography with nitrogen-phosphorus detection.

Toxicological evaluation of the antihistamines methapyrilene hydrochloride, pyrilamine maleate, and triprolidine hydrochloride monohydrate using methapyrilene hydrochloride as the positive indicator was investigated as part of a structure-activity relationship study in rats and mice. Prerequisites for the toxicological tests were the development of analytical procedures to certify the dose, homogeneity and stability of the drugs in animal feed and to monitor human urine for possible exposure and to ensure removal of the test agents from wastewater prior to its discharge into the environment. A high-performance liquid chromatographic (HPLC) system was developed using a fluorescence detector for the determination of methapyrilene hydrochloride and pyrilamine maleate in feed at levels as low as 100 ng/g and in human urine as low as 1 ng/g. An HPLC-UV procedure was developed for the determination of triprolidine hydrochloride monohydrate in feed at levels as low as 10 micrograms/g. Data concerning p-values, extraction efficiencies from feed and stability experiments in feed are presented for these antihistamines. A gas chromatographic procedure using a nitrogen-phosphorus detector was also developed for determining the three antihistamines in admixture in wastewater at levels as low as 10 ng/g.

High performance liquid chromatographic determination of methapyrilene hydrochloride in feed and sleep aid tablets.

Methapyrilene hydrochloride (MP-HCl) was extracted from feed with methanol and determined by reverse phase partition chromatography in less than 15 min, using isocratic elution with acetonitrile-1.1% ammonium carbonate (1 + 1) as the mobile phase. This procedure was tested on feed treated with MP-HCl at levels of 125, 500, and 2000 ppm. Recoveries were 104, 95, and 96% with coefficients of variation of 2.4, 1.6, and 0.6%, respectively. MP-HCl in feed was stable for 14 days. This method was also successfully used to determine MP-HCl in 3 sleep aid tablets.

In vitro nitrosation of methapyrilene.

The reactions of sodium nitrite and methapyrilene were studied in aqueous solution at neutral pH and under simulated gastric fluid conditions. Reaction product formation was much more complex than nitrosation of the parent molecule dimethylamino moiety to form nitrosodimethylamine. Several new nitroso compounds were formed under the reaction conditions studied. The simultaneous incorporation of 2 moles of ascorbic acid/mole of nitrite ion prevented any destruction of methapyrilene under all conditions studied. The implications of these observations with respect to nitrosation theory, the general carcinogenicity of nitroso compounds, and methapyrilene dosage formulation are discussed.

Simultaneous semiautomated assay of pyrrobutamine phosphate, cyclopentamine hydrochloride, and methapyrilene hydrochloride in pharmaceutical mixtures.

Antihistamine preparations containing methapyrilene hydrochloride, pyrrobutamine phosphate, and cyclopentamine hydrochloride were assayed by introducing an aqueous sample solution into the appropriate automated system. Methapyrilene hydrochloride was determined by UV spectrophotometry. Pyrrobutamine phosphate was extracted as an ion-pair and quantitated colorimetrically by forming the bromcresol purple acid-dye complex. Cyclopentamine hydrochloride was determined colorimetrically by using the copper dithiocarbamate reaction for secondary amines.

Comparative toxicology in vitreous humor and blood.

Drug and toxic substances were detected in blood and vitreous humor in fifty-six cases, in which causes of death were both from an overdose of the particular substances and from other unrelated causes. Five instances are reported in which two drug substances were detected in blood and vitreous humor from the same subject. Patients having long survival times, as well as those dying from unrelated causes, reveal drug values to approach unity, when the blood and vitreous concentrations are compared. The ratios reached at equilibrium probably depend on solubility of the drug in vitreous humor, lipid solubility and the percentage protein-bound in the blood. The vitreous humor provides another parameter of testing and may be useful in studies of survival time.