DL-α-difluoromethylornithine Research Articles

Contragestational effects of DL-α-difluoro-methylornithine, an irreversible inhibitor of ornithine decarboxylase, in the hamster

In the hamster, there are remarkable changes in ODC activity in various uterine reproductive tissues during gestation. When DL-α-difluoromethylornithine (DFMO), a highly selective irreversible inhibitor of ODC, is given during the early post-implantation period, when ODC activity in the conceptus is rapidly rising and reaches its highest values, it causes pregnancy arrest. Low doses of DFMO are contragestational only during a short period of gestation (day 7 and 8), with primary action on the embryo. Higher doses given soon after implantation (day 5) induce early pregnancy termination by a primary effect on decidual tissue. Biochemical investigations in animals treated on day 5 or day 7 showed that DFMO induces a rapid and dramatic fall in ODC activity in the products of conception. These findings strengthen and extend previous evidence that ODC and polyamines play an essential role in the maintenance of early pregnancy in rodents.

Inhibition of rat embryonic development by the intrauterine administration of α-difluoromethylornithine

Decarboxylation of L-ornithine by L-ornithine decarboxylase (ODC; E.C.4.1.1.17.) is the initial step in the biosynthesis of putrescine. ODC activity is generally low in most tissues, marked increases are associated with rapid tissue growth and particularly with mammalian embryogenesis. 0.5 mg/kg of DL-α-difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) was administered to uterine horns of Long-Evans adult rats during the 4th day of pregnancy. As control material, saline (0.15 M) was administered to contralateral uterine horn. The animals were sacrificed on different days, the uterine horns were removed and the number of implanted embryos were counted. DNA, RNA, protein and dry weight content in implantation sites (5th day of pregnancy) indicated that decidualization following DFMO took place normally but that embryonic growth was arrested in the treated horn. When 100μg of putrescine were added together with DFMO, the embryotoxic effect was absent.

Suppression of ribosomal gene expression in oocytes and nurse cells of a polychaete as a result of polyamine synthesis inhibition

The role of the polyamines in ribosomal gene expression was evaluated in the polychaete Ophryotrocha labronica by analyzing the effects of polyamine synthesis inhibition on RNA synthesis during oogenesis, a period characterized by intense nucleolar activity. At various stages of oogenesis adult polychaete females were blocked in their polyamine synthesis by the addition of 10 mM dl-α-difluoromethylornithine (DFMO) to the sea water in which they were cultivated. To monitor RNA synthesis during DFMO treatment the animals were pulse-labeled with [5- 3H]uridine and processed for autoradiography. Light and electron microscope autoradiographs demonstrate that DFMO treatment suppresses incorporation of label into nucleolar RNA (rRNA) both in the oocytes and their associated nurse cells. The ultrastructural appearance of both cell types reveals interference with nucleolar and ribosomal activity; the endoplasmic reticulum is deprived of ribosomes, and the production of protein granules (vitellogenesis) is reduced. The high specificity of DFMO for polyamine synthesis and the fact that the effects of DFMO were counteracted by addition of a low concentration (10 μM) of putrescine shows that the observed interference with ribosomal gene expression is indeed due to polyamine deficiency.

Treatment of metastatic lewis lung carcinoma with dl-α-difluoromethylornithine

The effects of dl-α-difluoromethylornithine (DFMO), a specific, irreversible inhibitor of ornithine decarboxylase (ODC), on tumors induced in the muscle of C57BL mice by Lewis lung (LL) carcinoma cells and on the development of lung metastases have been investigated. ODC activity and putrescine, spermidine and spermine concentrations were increased both during the early phase of development of the primary LL tumor and in the lung coinciding with the development of metastases. Oral treatment with DFMO (2% aqueous solution as sole drinking fluid, equivalent to 4 g DFMO/ kg/day) decreased markedly the ODC activity and the putrescine and spermidine concentrations of the primary tumor, and stimulated S-adenosyl- l -methionine decarboxylase activity. ODC activity and putrescine and spermidine concentrations were similarly markedly reduced in the metastatic lung by DFMO treatment. By comparison with untreated controls, DFMO treatment from day 1 after inoculation resulted in an 81% decrease in tumor size and a 92% reduction of lung metastases by day 20 and prolonged the mean survival time from 20.2 to 28.8 days. The same treatment regimen started 8 days after tumor inoculation resulted in a 52% inhibition of tumor growth and an 82% reduction of lung metastases, and prolonged the mean survival time to 24.9 days. The clear antitumoral effects obtained with DFMO on this animal metastatic cancer indicate its potential value in the treatment of metastases in humans.

Enhancement of ovulation in the rat by DL-α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase

Spontaneously cycling Sprague-Dawley Rats were treated with the irreversible ornithine decarboxylase (ODC) inhibitor DL-α-difluoromethylornithine (DFMO) on the day of proestrus. This treatment completely suppressed both the rise in ovarian ODC and the associated increase in putrescine concentration and increased the number of eggs released the next day by 91%. At the next oestrus (5 days after DFMO) there was an increase of 53% but at the third oestrus after DFMO (9 days) there was no significant change in the number of eggs released. A model is proposed in which ornithine decarboxylase forms part of a local inhibitory feed-back system controlling the number of follicles which develop to maturity and rupture during each cycle.

Dissociation of 12- O-tetradecanoylphorbol-13-acetate and 3-methylcholanthrene-induced induction in ornithine decarboxylase and aryl hydrocarbon hydroxylase activities in [formula omitted] mouse dermal fibroblasts in culture

The effects of 12- O-tetradecanoylphorbol-13-acetate (TPA) and 3-methylcholanthrene (MC) on ornithine decarboxylase (ODC) and aryl hydrocarbon hydroxylase (AHH) activities were studied in C57BL 6 mouse dermal fibroblasts in culture. TPA selectively induced ODC activity and MC selectively induced AHH activity in these cells. Acute (10 h) exposure of the cells to DL-α-difluoromethyl ornithine (DFMO) led to a marked inhibition of ODC activity without any significant effect on induced AHH activity. Chronic inhibition of ODC activity (4 days) resulted in a slight inhibition of basal AHH activity, but the inducibility of AHH was enhanced. α-Naphthoflavone, SKF 525 A and indomethacin exerted unequal effects on the enzyme activities. These results indicate that ODC and AHH induction processes are independent events with no causal link.

Assay of α-difluoromethylornithine in body fluids and tissues by automatic amino-acid analysis

A procedure is described for the measurement of dl-α-difluoromethylornithine (DFMO), a selective irreversible inhibitor of ornithine decarboxylase, in biological specimens. The drug is separated from other amino acids with a commercial amino-acid analyser and detected by formation of its alkylthio-isoindole derivative with o-phthalaldehyde. DFMO concentrations of 0.1 nmol can be determined in a sample volume of 100 μl. The assay has been used to determine the half-life of DFMO in serum of several species and the relationships between serum and tissue concentrations.

Depletion of 9L rat brain tumor cell polyamine content by treatment with d,l-α-difluoromethylornithine inhibits proliferation and the G1 to S transition

d,l-α-Difluoromethylornithine (DFMO), an irreversible inactivator of ornithine decarboxylase, inhibited 9L monolayer culture rat brain tumor cell proliferation at concentrations as low as 1 mM DFMO to about 25% of control growth when cells were seeded at an initial density of 5 × 10 5/flask. DFMO reduced intracellular putrescine content to <5% of control by 8 h and spermidine content to <5 % of control by 48 h post-treatment. Cytostasis caused by 10 or 25 mM DFMO could both be reversed and blocked by addition of exogenous putrescine. Cells pretreated for 48 h with DFMO and then replated in its absence could not enter exponential growth until polyamine production resumed. Addition of exogenous putrescine at the time of replating allowed pretreated cells to resume exponential growth at the same time as controls. Flow cytometry revealed that the fraction of cells in G1 increased until polyamine accumulation resumed, implying the presence of a G1-S block. Within 6 h of replating, there was a decrease in the fraction of control cells in G1. These observations support the hypothesis that entry of 9L cells into S phase depends on an adequate intracellular pool of polyamines.

Ovarian function in the rat following irreversible inhibition of L-ornithine decarboxylase

The possibility that the transient rise in rat ovarian ornithine decarboxylase (ODC) activity and the associated increase in putrescine which occur under luteinizing hormone control late in proestrus have an essential role in ovarian function has been tested using DL-α-difluoromethylornithine (DFMO) an irreversible inhibitor of ODC. Treatment with DFMO, 500 mg/kg s.c. at 12:00 h on the day of proestrus and 200 mg/kg, 6, 12 and 18 h thereafter completely suppressed both the rise in ovarian ODC activity and the associated increase in putrescine concentrations. However, ovulation took place normally under these conditions and the course of the resulting pregnancies was also normal. Similarly, combined treatment with DFMO and an inhibitor of S-adenosyl-L-methionine decarboxylase, 1,1-((methylethanediylidine)-dinitrilo) bis (3-aminoguanidine), 25 mg/kg, given at 10:00 h on the morning of proestrus failed to influence either ovulation or the subsequent period of gestation. These data provide no support for a functional role of the pre-ovulatory rise of ODC in rat ovary in the major peri-ovulatory events of that particular cycle, although they do not exclude effects on systems (e.g. steroidogenesis) not directly examined with our experimental approach. On the other hand, inhibition of ODC resulted in an increase in the number of uterine implantation sites following mating at the next proestrusestrus 4 days later. These data would support the previously expressed view that ODC may be associated with the gonadotrophin-induced initiation of follicular development for ovulation in the succeeding cycle. Moreover, since inhibition of the enzyme resulted in facilitation of the process, the normal physiological function of ODC, and/or the putrescine generated through its action, would appear to be inhibitory.