Evidence that GABAA receptor subunit mRNA expression during development is regulated by GABAA receptor stimulation.

The expression of six mRNA species (alpha 2, alpha 3, alpha 5, beta 2, beta 3, and gamma 2) encoding for GABAA receptor subunits was followed in cultured early postnatal cortical neurons by in situ hybridization histochemistry. In untreated control cultures it was found that these subunit mRNA expression profiles closely follow those seen during development in vivo. alpha 3, alpha 5, and beta 3 subunit expression declined, alpha 2 expression increased, whereas beta 2 and gamma 2 subunit mRNA expression remained relatively constant. To test the hypothesis that GABAA receptor stimulation regulates these expression profiles, we tested the effect of a GABAA receptor positive modulator, allopregnanolone, and a GABAA receptor noncompetitive antagonist, tert-butylbicyclophosphorothionate (TBPS). It was found that allopregnanolone augmented the rate at which the alpha 3, alpha 5, or beta 3 subunit mRNA expression declined and prevented the increase in alpha 2 subunit mRNA expression. As well, allopregnanolone down-regulated beta 2 subunit mRNA expression. TBPS, on the other hand, up-regulated alpha 3, alpha 5, beta 2, and beta 3 subunit mRNA expression. It also down-regulated the expression of alpha 2 subunit mRNA. Both allopregnanolone and TBPS had no effect on gamma 2 subunit mRNA expression. These results imply that the developmental switchover of GABA receptor subunit mRNA expression is regulated by GABAA receptor activity.

Deuterium isotope effect on the toxicokinetics of monomethylamine in the rat.

The single-dose toxicokinetics of monomethylamine has been characterized in the rat by HPLC assay of serial blood samples. Biphasic first-order elimination was observed following an iv bolus dose of 19 mumol/kg with a terminal half-life of 19.1 +/- 1.3 min (mean +/- SE, N = 4). The apparent steady state volume of distribution, systemic blood clearance, and renal blood clearance were 1.21 +/- 0.09 liter/kg, 53.4 +/- 3.5 ml/min/kg, and 5.72 +/- 0.53 ml/min/kg, respectively. The administration of an intragastric dose permitted the calculation of the systemic bioavailability of monomethylamine as 69 +/- 3%. Duplicate experiments using the structural analogue with deuterium atoms substituted for hydrogens on the methyl group revealed a much slower elimination of the compound, although ultimately, 5 times as much was excreted unchanged in the urine. Isotope effects calculated as the ratios of terminal half-life, systemic blood clearance, and systemic bioavailability were 1.9, 2.2, and 1.8, respectively.

Decomposition and quality control considerations in biological work with fecapentaene preparations.

Solutions of synthetic fecapentaene 12 (FP-12) intended for carcinogenicity studies were found to decompose extremely rapidly during customary dosage procedures. Apparent half-lives as short as 15 min were observed. While rates and even the qualitative course of decomposition were surprisingly variable in replicate experiments, high concentration and exposure to air were confirmed to be especially important destabilizing influences. The results suggested a primary role for a radical decomposition mechanism in the presence of atmospheric oxygen. Consistent with this hypothesis, FP-12 solutions were significantly stabilized by the radical chain-breaking antioxidant vitamin E. On the other hand, dithiothreitol greatly destabilized FP-12, presumably because of its nucleophilicity. The diacetyl diester of FP-12 was more soluble than the parent diol, but its decomposition rates in the presence and absence of vitamin E were similar to those of unesterified FP-12. Ultraviolet irradiation of an all-trans-FP-12 solution decreased its concentration by 70% in 0.5 min. The mutagenicities of the decomposition/isomerization products of FP-12, as studied in Salmonella typhimurium tester strain TA 100, ranged from negligible to comparable with all-trans-FP-12 itself. It is concluded that unchecked decomposition of fecapentaene preparations can profoundly affect biological tests therewith. While this can be largely controlled through the use of rigorous precautions, including protection from air, light, nucleophiles, and acids as well as selection of the lowest concentration compatible with the application at hand, the data argue strongly for inclusion of appropriate quality control measures in all future dosing operations to prove that the biological activity reported is that of the fecapentaene itself rather than that of a decomposed dosing solution.

Inactivity of fecapentaene-12 as a rodent carcinogen or tumor initiator.

The possible carcinogenic activity of synthetic fecapentaene-12 (FP-12) was studied in several mammalian test systems: (a) for carcinogenicity by intrarectal instillation in male F344/NCr rats as well as by intrarectal and subcutaneous application in male B6C3F1 mice; (b) for initiation by skin painting in female SENCAR mice followed by repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA), with 7,12-dimethylbenz[a]anthracene (DMBA) followed by TPA as positive control; (c) in a rat subcutaneous granuloma pouch assay in which mutagenicity was measured by induction of 6-thioguanine (6-TG) resistance and carcinogenicity was determined by induction of subcutaneous tumors in the pouch. There was no significant increase in tumor incidence after 72-78 weeks in test (a), although 2 rats receiving FP-12 intrarectally developed colon polyps. FP-12 did not initiate any skin tumors in test (b), nor did it significantly convert DMBA-initiated papillomas into carcinomas when 8 of the positive control mice were given FP-12 weekly for 10 weeks after 10 weeks on the DMBA-TPA regimen. Although FP-12 and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were comparably mutagenic in test (c), FP-12 induced no tumors after more than a year in 133 rats at risk while MNNG induced 7 tumors in 107 rats. These rodent assays provide no evidence that FP-12 is a strong carcinogen, although the possibility remains that it may possess weak carcinogenic activity not revealed by these experiments.