Some azo dyes, including Sudans I–IV and Para Red, are genotoxic and may be biotransformed to cancerogenic aromatic amines. They are banned as food and feed additives, but their presence has been detected in food. Aromatic amines are also considered potentially toxic. Online EC–MS is a promising tool to study the transformation mechanisms of xenobiotics such as azo dyes. The aim of the study was to investigate emulation of how azo dyes are enzymatically transformed to amines with EC–MS. The reduction reactions of five azo dyes (Sudans I–IV and Para Red) were conducted using a glassy carbon working electrode and 0.1% formic acid in acetonitrile. Reduction results were compared with the literature and in silico to select preliminary candidates for metabolites. The LC-MS/MS method was used to confirm results obtained by electrochemical reactor. A limited number of pre-selected compounds were confirmed as azo dyes metabolites – aniline for Sudan I, aniline and 4-aminoazobenzene for Sudan III, o-toluidine for Sudan IV, and 4-nitroaniline for Para Red. No metabolites were found for Sudan II. Electrochemistry–mass spectrometry was successfully applied to azo dyes. This approach may be used to mimic the metabolism of azo dyes, and therefore predict products of biotransformation.

Introduction: The cytotoxicity of anthelmintic agent, albendazole (ABZ) and its two major metabolites, sulfoxide (ABZSO) and sulfone (ABZ-SO₂), on non-hepatic Balb/c 3T3 line, two hepatoma cell lines (FaO, HepG2), and isolated rat hepatocytes was investigated. Material and Methods: Cell cultures were exposed for 24, 48, and 72 h to eight concentrations of the compounds ranging from 0.05 to 100 μg/mL (ABZ) and from 0.78 to 100 μg/mL (ABZ-SO and ABZ-SO₂). Three different assays were applied in which various biochemical endpoints were assessed: lysosomal activity - neutral red uptake (NRU) assay, proliferation - total protein contents (TPC) assay and lactate dehydrogenase (LDH) leakage assay. Results: The most toxic was albendazole whose EC₅₀ values calculated from the concentration effect curves ranged from 0.2 to 0.5 μg/mL (Balb/c 3T3) and from 0.4 to 73.3 μg/mL (HepG2). Rat hepatoma line and isolated rat hepatocytes were less sensitive to the impact of ABZ. Toxic action expressed as EC₅₀ was recorded after 72 h exposure only in LDH release assay at 0.8 μg/mL and 9.7 μg/mL respectively. The toxicity of metabolites was much lower. The most sensitive to ABZ-SO were fibroblasts and EC₅₀₋₇₂ₕ values were similar in all three assays used, i.e. NRU (14.1 μg/mL), TPC (15.8 μg/mL), and LDH (20.9 μg/mL). In the case of ABZ-SO₂ the mean effective concentrations were the highest, and could be reached only in one LDH assay. These values (μg/mL) were as follows: 65.3 (FaO), 65.4 (HepG2), 75.8 (hepatocytes), and 77.4 (Balb/c 3T3). Conclusion: The differences in in vitro toxicity of albendazole depend on metabolic ability of the cellular models. Primary cultured rat hepatocytes represent a valuable tool to study the impact of biotransformation on the cytotoxicity of drugs.

Objective: To determine plasma pharmacokinetics of penciclovir following oral and rectal administration of famciclovir to young Asian elephants (Elephas maximus). Animals: 6 healthy Asian elephants (5 females and 1 male), 4.5 to 9 years old and weighing 1,646 to 2,438 kg. Procedures: Famciclovir was administered orally or rectally in accordance with an incomplete crossover design. Three treatment groups, each comprising 4 elephants, received single doses of famciclovir (5 mg/kg, PO, or 5 or 15 mg/kg, rectally); there was a minimum 12-week washout period between subsequent famciclovir administrations. Serial blood samples were collected after each administration. Samples were analyzed for famciclovir and penciclovir with a validated liquid chromatography–mass spectroscopy assay. Results: Famciclovir was tolerated well for both routes of administration and underwent complete biotransformation to the active metabolite, penciclovir. Mean maximum plasma concentration of penciclovir was 1.3 μg/mL at 1.1 hours after oral administration of 5 mg/kg. Similar results were detected after rectal administration of 5 mg/kg. Mean maximum plasma concentration was 3.6 μg/mL at 0.66 hours after rectal administration of 15 mg/kg; this concentration was similar to results reported for humans receiving 7 mg/kg orally. Conclusions and Clinical Relevance: Juvenile Asian elephants are susceptible to elephant endotheliotropic herpesvirus. Although most infections are fatal, case reports indicate administration of famciclovir has been associated with survival of 3 elephants. In Asian elephants, a dose of 8 to 15 mg of famciclovir/kg given orally or rectally at least every 8 hours may result in penciclovir concentrations that are considered therapeutic in humans.

Objective—To identify and characterize cytochrome P450 enzymes (CYPs) responsible for the metabolism of racemic ketamine in 3 mammalian species in vitro by use of chemical inhibitors and antibodies. Sample—Human, canine, and equine liver microsomes and human single CYP3A4 and CYP2C9 and their canine orthologs. Procedures—Chemical inhibitors selective for human CYP enzymes and anti-CYP antibodies were incubated with racemic ketamine and liver microsomes or specific CYPs. Ketamine N-demethylation to norketamine was determined via enantioselective capillary electrophoresis. Results—The general CYP inhibitor 1-aminobenzotriazole almost completely blocked ketamine metabolism in human and canine liver microsomes but not in equine microsomes. Chemical inhibition of norketamine formation was dependent on inhibitor concentration in most circumstances. For all 3 species, inhibitors of CYP3A4, CYP2A6, CYP2C19, CYP2B6, and CYP2C9 diminished N-demethylation of ketamine. Anti-CYP3A4, anti-CYP2C9, and anti-CYP2B6 antibodies also inhibited ketamine N-demethylation. Chemical inhibition was strongest with inhibitors of CYP2A6 and CYP2C19 in canine and equine microsomes and with the CYP3A4 inhibitor in human microsomes. No significant contribution of CYP2D6 to ketamine biotransformation was observed. Although the human CYP2C9 inhibitor blocked ketamine N-demethylation completely in the canine ortholog CYP2C21, a strong inhibition was also obtained by the chemical inhibitors of CYP2C19 and CYP2B6. Ketamine N-demethylation was stereoselective in single human CYP3A4 and canine CYP2C21 enzymes. Conclusions and Clinical Relevance—Human-specific inhibitors of CYP2A6, CYP2C19, CYP3A4, CYP2B6, and CYP2C9 diminished ketamine N-demethylation in dogs and horses. To address drug-drug interactions in these animal species, investigations with single CYPs are needed.

Pharmacodynamic variables of enrofloxacin were investigated in a neutropenic mouse Escherichia coli and staphylococcal thigh infection model. Enrofloxacin pharmacokinetics in clinically normal mice and dogs were compared to confirm that doses evaluated in the mouse model would include enrofloxacin doses appropriate for use in dogs. Mice were made neutropenic by treatment with cyclophosphamide and injected in the thigh muscle with approximately 10(6) colony-forming units of E. coli (n = 2) or a staphylococcal (n = 2) clinical isolate. Enrofloxacin dosages tested ranged from 0.78 to 50 mg/kg of body weight and 6.25 to 200 mg/kg in the E. coli and staphylococcal infection trials, respectively. In each 24-hour dosage trial, enrofloxacin was administered SC as a single dose or in divided doses given every 3, 6, or 12 hours. Comparison of log(10) colony-forming units per thigh muscle in untreated control mice and mice treated with enrofloxacin was used as a measure of efficacy. Two-way ANOVA was used to determine that the enrofloxacin total dose, but not the dose frequency, was significant in determining drug efficacy. Pharmacokinetic values analyzed by use of multivariant stepwise linear regression analysis indicated that the area under the concentration-time curve, but not time above minimum inhibitory concentration, was significant in predicting efficacy of enrofloxacin treatment. We conclude that enrofloxacin killing of E. coli and staphylococci is concentration dependent and not time dependent.