Dose-response analysis of epigenetic, metabolic, and apical endpoints after short-term exposure to experimental hepatotoxicants.

Identification of sensitive and novel biomarkers or endpoints associated with toxicity and carcinogenesis is of a high priority. There is increasing interest in the incorporation of epigenetic and metabolic biomarkers to complement apical data; however, a number of questions, including the tissue specificity, dose-response patterns, early detection of those endpoints, and the added value need to be addressed. In this study, we investigated the dose-response relationship between apical, epigenetic, and metabolomics endpoints following short-term exposure to experimental hepatotoxicants, clofibrate (CF) and phenobarbital (PB). Male F344 rats were exposed to PB (0, 5, 25, and 100 mg/kg/day) or CF (0, 10, 50, and 250 mg/kg/day) for seven days. Exposure to PB or CF resulted in dose-dependent increases in relative liver weights, hepatocellular hypertrophy and proliferation, and increases in Cyp2b1 and Cyp4a1 transcripts. These changes were associated with altered histone modifications within the regulatory units of cytochrome genes, LINE-1 DNA hypomethylation, and altered microRNA profiles. Metabolomics data indicated alterations in the metabolism of bile acids. This study provides the first comprehensive analysis of the apical, epigenetic and metabolic alterations, and suggests that the latter two occur within or near the dose response curve of apical endpoint alterations following exposure to experimental hepatotoxicants.

Occurrence, fate and effects of pharmaceutical substances in the environment--a review.

Medical substances (pharmaceuticals) are a group of substances that until recently have been exposed to the environment with very little attention. The reason why they may be interesting as environmental micropollutants, is that medical substances are developed with the intention of performing a biological effect. Especially antibiotics used as growth promoters, as feed additives in fish farms are anticipated to end up in the environment. Very little is known about the exposure routes of the medical substances to the environment. Only few investigations have reported findings of medical substances in other field samples than sediment or treated waste water samples. Several substances seem to be persistent in the environment. This paper outlines the different anticipated exposure routes to the environment, summarises the legislation on the subject and gives an outline of present knowledge of occurrence, fate and effect on both the aquatic and terrestrial environments of medical substances. Present knowledge does not reveal if regular therapeutic use may be the source of a substance carried by sewage effluent into the aquatic system, even though clofibrate, a lipid lowering agent, has been identified in ground and tap water samples from Berlin. Further research would be necessary to assess the environmental risk involved in exposing medical substances and metabolites to the environment.

Preparative chromatography of furosemide 1-O-acyl-glucuronide from urine using micronized amberiite XAD-2 and its application to other 1-O-acyl-glucuronides.

Furosemide 1-O-acyl glucuronide (Fgnd) was extracted from the urine following oral administration of furosemide. The crude Fgnd was applied to micronized Amberlite XAD-2 column (2.5 cm i.d. x 90 cm length, 75-500 microns particle size). The purified Fgnd was identified by mass spectrometry and beta-glucuronidase treatment. This method was also applicable to the purification of glucuronide of tolmetin (nonsteroidal anti-inflammatory drug, NSAID), suggesting that it was applicable to the other NSAIDs, most of which were known to be metabolized to acyl-glucuronides.

Pharmacokinetic studies with the lipid-regulating agent beclobrate: enantiospecific assay for beclobric acid using a new fluorescent chiral coupling component (S-FLOPA).

The major biotransformation pathway for the chiral lipid-regulating agent beclobrate is conversion to the corresponding carboxylic acid, which is then metabolized to the acyl glucuronide. An enantiospecific assay for biological material was developed that is based on chiral derivatization with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide (EDAC) and the primary amine S-FLOPA, a new chiral coupling component for carboxylic acids derived from the 2-arylpropionic acid S-flunoxaprofen. Conversion of beclobric acid to the acyl chloride prior to coupling with the amine is also feasible. From plasma or urine beclobric acid was extracted into n-hexane/ethanol (9:1) at pH 4 after addition of sodium chloride. Clofibric acid was used as internal standard. Derivatization with EDAC/FLOPA was performed under addition of 1-hydroxybenzotriazole in anhydrous dichloromethane containing trace amounts of pyridine (ambient temperature/2 h reaction time). The chromatographic separation was performed on a silica gel stationary phase (Zorbax Sil) using n-hexane-chloroform-ethanol (100:10:0.75, by vol) as mobile phase [flow rate, 2 ml/min; fluorescence detection, 305/355 nm; elution order of the derivatives, (-) before (+)]. Coefficients of variation were between 1.3 and 9.3% for both plasma and urine. Limit of quantification was 20-25 ng/ml for plasma based on a sample volume of 0.2 ml. Application of the assay in a pilot pharmacokinetic study showed significant differences between the kinetics of the two enantiomers. In plasma and urine, the concentrations of the dextrorotatory enantiomer exceeded those of the levorotatory enantiomer significantly.

In vitro dissolution profile of water-insoluble drug dosage forms in the presence of surfactants.

The determination of the in vitro release profile of water-insoluble drug products requires dissolution media different from those used for water-soluble drug products. Since the relevance of drug dissolution in organic solvents is questionable, we investigated the use of surfactants to determine the dissolution profiles of water-insoluble drug products. In most cases, the drug dissolution rate and extent increased as the surfactant concentration in the aqueous dissolution medium increased. Suitable dissolution profiles were obtained in the presence of sodium lauryl sulfate (SLS) for water-insoluble drug products, such as griseofulvin, carbamazepine, clofibrate, medroxyprogesterone, and cortisone acetate. These findings recommend the use of surfactants for determining the aqueous dissolution of water-insoluble drug products rather than adding organic solvents to the dissolution medium.

Clofibrate, caloric restriction, supersaturation of bile, and cholesterol crystals.

Lipid composition, cholesterol saturation, and cholesterol crystal formation of gallbladder bile were studied in seven type-IV hyperlipoproteinemic subjects who did not have gallstones. Thereafter, biliary cholesterol solubilization was overloaded, first by clofibrate and then by caloric restriction treatment. Initially increased cholesterol saturation was still increased by both clofibrate and caloric restriction treatment, but none of the subjects developed cholesterol crystals in bile, indicating that they had a mechanism to maintain cholesterol in solution in the bile despite remarkable supersaturation. This suggests that the patients who are at risk of developing gallstones can be better selected by cholesterol crystal analysis of bile samples than by analysis of lipid composition of bile.

Determination of tibric acid and clofibrate in animal feed, wastewater and hunan urine.

Analytical chemical procedures are described to determine residues of the drugs clofibrate and tibric acid in animal feed, wastewater, and human urine. Clofibrate was extracted from animal feed and human urine with hexane, whereas residues from wastewater were collected on a Sep-PakTM then eluted with methanol for analysis. Clofibrate residues from the feed, wastewater, and urine were analyzed by high-pressure liquid chromatography (HPLC) with minimum detectable levels (MDL) of about 40, 0.5 and 1.0 ppb, respectively. Tibric acid was extracted from animal feed with 90% methanol and 10% 0.1 N NaOH, whereas wastewater and human urine were acidified with 12 N HCl and then extracted with benzene. The MDL for tibric acid in feed by electron capture/gas chromatography (EC/GC) and HPLC were about 40 ppb and 2.0 ppm, respectively. Residues from these extracts that contained more than 5 ppm of tibric acid were analyzed by HPLC, whereas GC was required for levels below 5 ppm. The GC procedures, which required that tibric acid be derivatized (methylated) prior to analysis, had MDL of 0.1 and 1.0 ppb for wastewater and human urine, respectively. Data are also presented concerning partition values, stability of the compounds in animal feed, and recoveries of the compounds from the three substrates.

High-pressure liquid chromatographic analysis of drugs in biological fluids. IV. Determination of clofibrinic acid.

A rapid, sensitive and specific high-pressure liquid chromatographic method is described for the quantitative analysis of clofibrinic acid in plasma, saliva and urine. In contrast to previously reported gas-liquid chromatographic methods, which require derivatization of clofibrinic acid before chromatography, the present method involves a simple two-step extraction procedure and chromatographic determination of the underivatized clofibrinic acid. Concentrations between 1.0 and 25.0 microgram per sample can be measured with a coefficient of variation from 1 to 6%.

Determination of clofibrate in biological fluids by thin-layer and gas-liquid chromatography.

A specific and sensitive method is described for the detection of clofibrate in biological fluids. The drug is separated from associated fatty acids by thin-layer chromatography and the methyl ester is quantified by gas-liquid chromatography. Recovery is excellent, and any small losses are corrected with an internal recovery standard. Although more time-consuming than other available techniques, the method offers advantages for accurate studies of clofibrate metabolism.