Primary rat hepatocytes as in vitro system for gene expression studies: comparison of sandwich, Matrigel and 2D cultures.

Recent studies have presented evidence that in vivo obtained gene expression data can be used for carcinogen classification, for instance to differentiate between genotoxic and non-genotoxic carcinogens. However, although primary rat hepatocytes represent a well-established in vitro system for drug metabolism and enzyme induction, they have not yet been systematically optimized for toxicogenomic studies. The latter may be confounded by the fact that cultured hepatocytes show strong spontaneous alterations in gene expression patterns. Therefore, we addressed the following questions: (1) which culture system is optimal, comparing sandwich, Matrigel and 2D cultures, (2) how critical is the impact of culture period on substance-induced alterations in gene expression and (3) do these substance-induced alterations in cultured hepatocytes occur already at in vivo relevant concentrations? For this purpose we analyzed the expression of four genes, namely Abat, Gsk3beta, Myd116 and Sult1a1 that recently have been reported to be influenced by the antihistamine and non-genotoxic carcinogen methapyrilene (MPy). The most reproducible effects of MPy were observed in sandwich cultures. Induction factors of Gsk3beta and Myd116 at 100 microM MPy were 2 and 4 (medians), respectively, whereas expression of Abat and Sult1a1 were inhibited by factors of 7 and 5, respectively. Similar results were observed in hepatocytes maintained for 24 h or 3 weeks in sandwich culture with respect to the influence of MPy on the expression of Abat, Gsk3beta, Myd116 and Sult1a1. To determine whether MPy influences gene expression at in vivo relevant concentrations, 3.5 mg/kg MPy were administered to male Wistar rats intraperitoneally, resulting in plasma concentrations ranging between 1.72 and 0.32 microM 5 and 80 min after injection. Inhibition of Abat and Sult1a1 expression in vitro already occurred at in vivo relevant concentrations of 0.39 microM MPy. Induction of Myd116 was observed at 6.25 microM which is higher but in the same order of magnitude as in vivo relevant concentrations. In conclusion, the presented data strongly suggest that sandwich cultures are most adequate for detection of MPy-induced gene expression alterations and the effect of MPy was detected at in vivo relevant concentrations.

Plasma elimination and urinary excretion of methapyrilene in the rat.

The metabolism and elimination of methapyrilene (2-[(2-dimethylaminoethyl)-2-thenylamino]pyridine) were characterized after the iv administration of 0.7 mg/kg or 3.5 mg/kg methapyrilene HCl plus [14C]methapyrilene HCl to adult male Fischer-344 rats. Approximately 40% and 35% of the administered dose was excreted in the urine in the first 24 hr in the low and high dose groups, respectively, as determined by liquid scintillation spectrophotometry. Fecal excretion accounted for 38% and 44% of the administered dose in the first 24 hr in the low and high dose groups, respectively, as confirmed via combustion analysis. The 24-hr urinary metabolic products consisted of one major and five minor radiolabeled compounds. The major metabolite was isolated with reversed-phase HPLC and identified as methapyrilene N-oxide. This was accomplished by comparison of the chromatographic and mass spectral characteristics of this metabolite with that of authentic methapyrilene N-oxide. Methapyrilene and mono-N-desmethyl methapyrilene also were identified after isolation with reversed-phase HPLC and comparison of their mass spectral and/or chromatographic properties with those of authentic compounds. The plasma metabolic profile was essentially the same as the urinary profile. The elimination of methapyrilene from plasma occurred through a first-order process. The terminal plasma elimination t1/2 of methapyrilene did not increase with increasing doses (2.75 hr, 0.7 mg/kg; 2.81 hr, 3.5 mg/kg); thus, methapyrilene does not exhibit dose-dependent elimination over this 5-fold dose range.

Methapyrilene kinetics and dynamics.

A study was undertaken to characterize the H1 receptor blockade, central nervous system depressant properties, and kinetic parameters of methapyrilene in man. Eight healthy subjects received, in random order at weekly intervals, placebo and methapyrilene 20 mg intravenously and 50 mg and 25 mg orally. Methapyrilene exhibited a moderate antihistaminic effect as measured by the reduction of histamine-provoked skin wheals. Sedation and drowsiness were detected only at the first sampling time (0.75 hr) after intravenous doses. The terminal plasma half-life ranged from 1.1 to 2.1 hr, apparent volume of distribution from 2.14 to 6.61 1/kg, and plasma clearance from 0.013 to 0.048 1/min/kg. Systemic bioavailability was low and showed large interindividual differences, ranging from 4% to 46%. Recovery of unchanged drug from the 24-hr urine was under 2% of the doses.

A fatality involving methapyrilene.

Tissue concentrations of methapyrilene are given in a fatal case. There are no indications that methapyrilene had any synergistic or antagonistic effect with the patient's cardiopulmonary condition, although when taken in high overdosages the pharmacologic effects may be altered. This case was supported by other data in an attempt to elucidate as many contributing factors as possible in evaluating lethal levels.

Methapyrilene toxicity.

Seven cases of drug overdosage involving methapyrilene have been presented, five of which resulted in death. Methapyrilene blood levels ranged fron 1.2 to 3.0 mg% (Table 2). Five of the seven cases involved multiple drug dosage with ethanol, salicylamide, amobarbital, secobarbital, and/or scopolamine. Of the remaining cases, involving only methapyrilene, ome fatality occurred at a blood level of 2.7 mg%. The surviving case involved the reported ingestion of 100 tablets of Sleep-eze (2.5 gm methapyrilene), wherein serial lavage removed 1.1 gm of methapyrilene. Urinalysis revealed 2.52 mg% of methapyrilene in 1300 ml of urine. The methapyrilene blood level was too low to quantitate.