Effect Of Difluoromethylornithine Research Articles

Effects of Difluoromethylornithine on Growth Inhibition and Apoptosis in Human Cervical Epithelial and Cancerous Cell Lines

Difluoromethylornithine(DFMO), an irreversible inhibitor of ornithine decarboxylase and an angiogenesis inhibitor, has been used in phase I cervical intraepithelial neoplasia (CIN) trials, producing a 50% regression of CIN 3 lesions. DFMO is currently in phase II trials. In the experiments reported here, DFMO's growth inhibition and apoptosis induction were explored in an in vitro model to elucidate mechanisms of action. Four immortalized cervical epithelial cell lines, serving as in vitro models of precancerous CIN lesions, and nine cervical carcinoma cell lines were studied. DFMO's growth inhibitory effect was tested in monolayer culture and in semisolid medium, and concentrations required for a 50% growth inhibition (IC(50)) with a 5-day treatment were determined. Apoptosis induction was analyzed using the terminal deoxynucleotidyl transferase assay of DNA fragmentation. DFMO inhibited growth of immortalized cervical epithelial cell lines and cervical cancer cell lines in monolayer culture and in semisolid medium. The immortalized cervical epithelial cell lines were more sensitive than the cervical cancer cell lines to DFMO's growth inhibitory effect. Concentrations required for 50% growth inhibition after a 5-day treatment ranged from 100 microM to >5 mM for cervical carcinoma cell lines and from 100 microM to 1 mM for immortalized cervical epithelial cell lines. DFMO induced apoptosis in precancerous and cancerous cell lines at a concentration of 5 mM, regardless of the cells' human papillomavirus status. DFMO inhibits the growth of cervical precancerous and cancerous cells in vitro in a dose-dependent and time-dependent manner, partially through inducing apoptosis.

Polyamine-fas Interactions: Inhibition of Polyamine Biosynthesis in MRL-lpr/lpr Mice is Associated with the Up-regulation of fas mRNA in Thymocytes

MRL-lpr/lpr is a strain of mice that develops spontaneous signs of the autoimmune disease, systemic lupus erythematosus (SLE or lupus). The lpr (lymphoproliferation) defect has been identified as an insertion of an early transposon (ETn) derived sequence into the fas apoptosis gene. We studied the in vivo effects of difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme, ornithine decarhoxylase (ODC), on the expression of fas in MRL-lpr/lpr mice as well as in congenic MRL-+/+ and autoimmune NZB/W strains. Using Northern blot hybridization and reverse transcription polymerase chain reaction (RT-PCR), we found that DFMO treatment resulted in an increase in the expression of fas mRNA in the thymus of MRL-lpr/pr mice. Using RT-PCR, we further found that the increased expression of fas was associated with the suppression of chimeric ETn/fas mRNA. With fractionated CD4+ and CD8+ T cells, we found a cell-specific effect of DFMO on chimeric ETn/fas expression in CD8+ cells. ETn/fas expression was detected in CD8 + T cells from untreated mice, but it was eliminated after DFMO treatment. HPLC analysis of polyamines showed depletion of putrescine and partial reduction of spermidine (35%) in DFMO-treated mice compared to controls. These results indicate that DFMO-mediated polyamine depletion is linked to the regulation of fas and chimeric ETn/fas in MRL-lpr/lpr mice. Elevated levels of polyamines in this strain, as found in earlier studies, may be associated with the progression of the autoimmune disease by altering the expression of fas gene or by facilitating the expression of chimeric ETn/fas. Our data also provide new mechanistic insights into the beneficial effects of DFMO on these mice.

Effect of inhibition of ornithine decarboxylase activity in a model of acute hepatocellular necrosis.

The effect of blockade of the enzyme ornithine decarboxylase by difluoromethylornithine (DFMO) on hepatocellular necrosis and survival in rats treated with thioacetamide (TAA) was investigated. In one experiment, the effect of DFMO on survival of rats with TAA-induced acute hepatocellular necrosis was determined. In another experiment, blood and liver specimens were obtained from DFMO or saline-treated rats 24 h after the administration of TAA for determinations of serum alanine aminotransferase (ALT) and liver content of polyamines and microsomal cytochrome P-450 and for assessment of hepatic histology. Liver polyamines were determined by reversed-phase HPLC and microsomal cytochrome P-450 content by dithionite-difference spectroscopy of CO-treated homogenates. The severity of hepatocellular necrosis was scored blindly. TAA-treated rats that received DFMO survived longer than saline-treated controls (P < 0.01). Serum ALT and liver putrescine concentrations were lower and the histological severity of acute hepatocellular necrosis was less in DFMO-treated rats with TAA-induced hepatocellular necrosis than in saline-treated controls (P < 0.05, P < 0.01 and P < 0.05, respectively). Total cytochrome P-450 levels were similar in DFMO and saline-treated rats with TAA-induced hepatocellular necrosis. DFMO increases survival in TAA-induced fulminant hepatic failure by decreasing the severity of acute hepatocellular necrosis. The beneficial effects of DFMO do not appear to be mediated by its effects on polyamine metabolism, but may be attributable to an effect of DFMO on thioacetamide metabolism or on an alternative pathway of ornithine metabolism.

2,4‐Dichlorophenoxy acetic acid and indoleacetic acid partially counteract inhibition of organogenesis by difluoromethylornithine

Difluoromethylornithine (DFMO) is a well known inhibitor of putrescine biosynthesis that has been reported to interact in varying ways with auxins such as indoleacetic acid (IAA) and 2,4‐dichlorophenoxy acetic acid (2,4‐D). In the present report DFMO is shown to inhibit root formation in isolated hypocotyl segments of Euphorbia esula L. (leafy spurge) grown in the dark on solidified nutrient media in Petri dishes. Shoot formation was only slightly inhibited by DFMO and only on media with salts and vitamins diluted 10‐fold. 2,4‐D inhibited both root and shoot formation in full strength or diluted media. Simultaneous application of both compounds resulted in partial reversal of root inhibition, but only at 450 nM 2,4‐D, the highest concentration used. In both media IAA also partially reversed DFMO effects on root formation. The effects of DFMO, 2,4‐D or IAA on root (or shoot) formation do not appear to be closely related to endogenous content of the polyamines determined by high performance liquid chromatography.

2,4-Dichlorophenoxy acetic acid and indoleacetic acid partially counteract inhibition of organogenesis by difluoromethylornithine

Difluoromethylornithine (DFMO) is a well known inhibitor of putrescine biosynthesis that has been reported to interact in varying ways with auxins such as indoleacetic acid (IAA) and 2,4-dichlorophenoxy acetic acid (2,4-D). In the present report DFMO is shown to inhibit root formation in isolated hypocotyl segments of Euphorbia esula L. (leafy spurge) grown in the dark on solidified nutrient media in Petri dishes. Shoot formation was only slightly inhibited by DFMO and only on media with salts and vitamins diluted 10-fold. 2,4-D inhibited both root and shoot formation in full strength or diluted media. Simultaneous application of both compounds resulted in partial reversal of root inhibition, but only at 450 nM 2,4-D, the highest concentration used. In both media IAA also partially reversed DFMO effects on root formation. The effects of DFMO, 2,4-D or IAA on root (or shoot) formation do not appear to be closely related to endogenous content of the polyamines determined by high performance liquid chromatography.

Dietary polyamines promote the growth of azoxymethane-induced aberrant crypt foci in rat colon.

We have examined whether dietary polyamines influence the formation and initial growth of azoxymethane (AOM)-induced aberrant crypt foci (ACF) in rat colon. Effects of a combination of dietary polyamines at three dose levels (putrescine: 50, 280, 740 nmol/g; spermidine: 10, 261, 763 nmol/g; spermine: 1, 31, 91 nmol/g) in the polyamine-poor AIN-76A diet were studied in animals in two different experimental situations: animals treated with AOM alone and animals treated with AOM + difluoromethylornithine (DFMO), a specific inhibitor of endogenous polyamine synthesis. In both experimental situations, dietary polyamines enhanced the growth of ACF, expressed as the number of large ACF (foci with three or more aberrant crypts, ACF > or = 3), whereas the formation of ACF, expressed as the number of ACF, was apparently not altered. In animals treated with AOM alone, maximal growth enhancing effect on ACF was nearly obtained with the median level of dietary polyamine. In rats fed a low polyamine diet, basic AIN-76A, DFMO reduced the growth of AOM-induced ACF by 83%. This inhibitory effect of DFMO was counteracted by dietary polyamines in a dose-dependent manner, and it was abolished at the highest level of polyamines. In conclusion, it was demonstrated that dietary polyamines are able to enhance the growth of AOM-induced ACF. Further, dietary polyamines reversed the DFMO-caused inhibition of ACF growth, probably by compensating for the DFMO-reduced endogenous polyamine synthesis.

Chemopreventive effect of difluoromethylornithine (DMFO) on mouse skin squamous cell carcinomas induced by Benzo(a)pyrene

Chemopreventive effect of difluoromethylornithine (DMFO) on mouse skin squamous cell carcinomas induced by Benzo(a)pyrene

Effects of inhibitors of deoxyhypusine synthase on the differentiation of mouse neuroblastoma and erythroleukemia cells.

Deoxyhpusine synthase catalyzes the conversion of lysine to deoxyhypusine residue on the eukaryotic initiation factor 5A (eIF-5A) precursor using spermidine as the substrate. Subsequent hydroxylation of the deoxyhypusine residue completes hypusine formation on eIF-5A. Polyamines (putrescine, spermidine, and spermine) have been implicated in tumor growth and differentiation. Because deoxyhypusine/hypusine formation is one of the most specific polyamine-dependent biochemical events, we decided to use N1-guanyl-1,7-diaminoheptane (GC7), a potent inhibitor for deoxyhypusine synthase, to assess the role of hypusine formation on tumor growth and differentiation. GC7 suppressed the growth of N2a mouse neuroblastoma cells and DS19 murine erythroleukemia cells at micromolar concentrations. However, within a narrow concentration range, GC7 could promote the differentiation of mouse neuroblastoma cells in the presence of suboptimal amount of dibutyryl cAMP. In contrast, GC7 blocked the differentiation of DS19 cells induced with hexamethylene bisacetamide. Polyamine depletion by difluoromethyl ornithine (DFMO) has previously been shown to promote differentiation of neuroblastoma cells but inhibits erythrodifferentiation. Since our studies demonstrated that GC7 mimics the action of DFMO on tumor differentiation, it is likely that the effect of DFMO on tumor differentiation is mediated by hypusine formation and that GC7 represents a more specific inhibitor that can alter the differentiation program in certain tumor cells.

Putrescine modulation of depolarization-induced [ 3H]serotonin release from fish brain synaptosomes

In this study, we investigated the effects of putrescine, a precursor of polyamine metabolism, on depolarization (potassium chloride, KCl)-induced [ 3H]serotonin (5-hydroxytryptamine (5-HT) release from brain synaptosomes of rainbow trout ( Oncorhynchus mykiss). An irreversible inhibitor of polyamine metabolism, difluoromethyl-ornithine (DFMO), inhibited the KCl-induced [ 3H]serotonin efflux, and addition of putrescine (PUT) reversed the inhibitory effects of the former in synaptosomal preparations. In Fura-2/AM loaded synaptosomes, KCl stimulated Ca 2+ influx. Hence, prior addition of EGTA abolished the KCl-induced Ca 2+ responses, indicating that KCl-induced depolarization recruited Ca 2+ from extracellular medium. The DFMO curtailed the KCl-evoked Ca 2+ influx, and addition of PUT reversed the inhibitory effects of DFMO. Furthermore, fish brain was found to contain polyamines in high amounts (e.g. putrescine from 600 ± 20 to 850 ± 30 nmol/g protein). These results suggest that polyamine putrescine, present in fish brain, might be involved in [ 3H]serotonin efflux via Ca 2+ influx mechanisms.

Difluoromethylornithine counteracts lesion-induced astrogliosis in rat hippocampus: enhancement of inhibitory effect by combined treatment with GM1 ganglioside.

The effect of difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase (ODC), the rate limiting enzyme of polyamine biosynthesis, and its combined action with GM1 ganglioside, was studied on the GFAP content in a model of remote astrogliosis evoked in the hippocampus by lateral fimbria transection. DFMO markedly suppressed hippocampal gliosis as measured by GFAP immunoblotting seven days postsurgery. Combined treatment with DFMO and GM1 ganglioside--a substance which alone also counteracts hippocampal gliosis, produced a stronger suppressive effect than DFMO. The results support the hypothesis of a causal link between lesion induced events: polyamine biosynthesis and astroglial reaction. Potentiation of the inhibitory effect of DFMO by GM1 ganglioside suggests that the latter does not act through the mechanism involving ODC suppression.

Effect of polyamines and polyamine biosynthesis inhibitors on spore germination and hyphal growth of Glomus mosseae

The main free polyamines identified in ungerminated spores of Glomus mosseae were putrescine and spermidine. Changes in the level of these metabolites occurred during dormancy break induced by a cold treatment. Exogenous application of these polyamines to spores after a dormancy break led to a marked increase in hyphal growth, suggesting that endogenous concentrations of putrescine and spermidine could be growth limiting factors. When polyamine biosynthesis via ornithine decarboxylase (ODC) was blocked by DFMO (α- dl -difluoromethylornithine), a specific irreversible inhibitor of ODC, spore germination and hyphal growth were greatly reduced. These effects were DFMO concentration-dependent. Simultaneous treatment with DFMO and putrescine attenuated the inhibitory effect of DFMO on hyphal growth indicating that polyamines may be involved in regulating hyphal growth of G. mosseae . In contrast, DFMA (α- dl -difluoromethylarginine), a specific irreversible inhibitor of arginine decarboxylase (ADC), the other pathway for polyamine biosynthesis, promoted hyphal growth. These results suggest that in G. mosseae , polyamine biosynthesis during hyphal growth is regulated by ODC. Both DFMO and DFMA have been shown to provide control of a number of plant diseases caused by phytopathogenic fungi, but only the use of the latter appears compatible with the development of a symbiotic endomycorrhizal fungus.

Putrescine export from leaves in relation to floral transition in Sinapis alba

Sinapis alba L. was induced to flower by either a long day or a displaced short day. Following collection of leaf (phloem) and root (xylem) exudates from induced and non‐induced plants, polyamines in the exudates were extracted, separated and analyzed quantitatively. The titers of free and conjugated putrescine the major polyamine fractions in all samples, increased early and markedly in leaf exudates during the floral transition, coinciding closely with movement of the floral stimulus out of the induced leaf. By contrast, putrescine titer in the root exudate did not increase. A spray of difluoromethylornithine (DFMO), an irreversible inhibitor of the putrescine‐biosynthetic enzyme ornithine decarboxylase, at hour 8 of the long day considerably reduced the titer of free and conjugated putrescine in leaf exudates, and at the same time, markedly decreased the flowering response of induced plants. This effect of DFMO on flowering was substantially reversed by a simultaneous application of putrescine to the roots. DFMO sprayed on induced plants also suppressed early activation of indices of both mitosis and DNA synthesis in the shoot apical meristem. These results support the view that the extra putrescine synthesized in induced leaves is a necessary component of the floral stimulus in Sinapis.

Modulation of ricin toxicity in mice by biologically active substances.

Ricin is a highly toxic protein produced by the castor bean (Ricinus communis). It is one of various protein toxins that consist of two subunits joined by a disulfide bridge. One chain facilitates entry of the toxin into the cell while the other chain exhibits RNA N-glycosidase activity, which attacks a specific site on 28S rRNA, preventing polypeptide elongation and leading to cell death. Although ricin and other protein toxins are potential health hazards, no antidote against these toxins exists. Thus, a number of selected compounds were screened for their ability to alter ricin lethality in mice, based on percentage survival and time to death following a ricin LD100 of 25 micrograms kg-1 i.p. While no compound tested prevented lethality, dexamethasone and difluoromethylornithine (DFMO) significantly extended survival time. The effects of DFMO on ricin toxicity were markedly influenced by altering various pharmacokinetic parameters. The antioxidants butylated hydroxyanisole and vitamin E succinate also extended survival time in response to a lethal dose of ricin, but to a lesser extent than did dexamethasone and DFMO. The Golgi apparatus inhibitors monensin, swainsonine and tunicamycin enhanced ricin toxicity, as evidenced by shortened survival times. In addition, various nucleoside analogs, including acyclovir and trifluridine as well as adenosine, guanosine and dibutyryl cyclic AMP, also potentiated the toxicity of ricin. The results demonstrate that the toxicity of ricin is modulated by a wide variety of structurally distinct chemicals and may involve different mechanisms. Furthermore, the extent and direction of the modulation of ricin toxicity is highly dependent upon pharmacokinetic variables, including dose and dosing interval.

Estradiol control of ornithine decarboxylase mRNA, enzyme activity, and polyamine levels in MCF-7 breast cancer cells: therapeutic implications.

Previous studies have shown that natural polyamines-putrescine, spermidine, and spermine-play a key role in the mechanism of action of estrogens in breast cancer. Ornithine decarboxylase (ODC) is the first enzyme of the polyamine biosynthetic pathway. To examine estrogenic regulation of polyamine biosynthesis in breast cancer, we measured ODC mRNA, ODC activity, and polyamine levels in G1 synchronized MCF-7 cells. ODC mRNA and activity increased four-fold over that of cells in G1 phase between 8 to 16 h after the addition of estradiol. Polyamine levels showed a sharp increase by 8 h after the addition of estradiol and decreased by 12 h. We further examined whether synthetic homologs of putrescine or spermidine could replace natural polyamines in supporting MCF-7 cell growth. Treatment of MCF-7 cells with 1 mM difluoromethylornithine (DFMO), an inhibitor of ODC, suppressed putrescine, spermidine, and spermine levels by 74, 78, and 10%, respectively, within 48 h. Cells treated with DFMO for 48 h were supplemented with either putrescine or its homologs or spermidine or its homologs. Diaminopropane, diaminobutane (putrescine), and diaminopentane were capable of fully or partially reversing the growth inhibitory effects of DFMO, whereas diaminoethane had no significant effect. Among a series of triamines, H2N(CH2)nNH(CH2)3NH2 (where n = 2 to 8; abbreviated as APn n = 4 for spermidine, or AP4), spermidine was most effective in reversing the effects of DFMO, whereas compounds with shorter or longer methylene bridging regions were less effective. AP8 was ineffective in reversing the growth inhibitory effects of DFMO. At 10 microM concentration, AP8 also inhibited DNA synthesis by 66%, as measured by [3H]-thymidine incorporation. These data show that MCF-7 cells have a strong requirement for polyamines for their growth and that estradiol stimulates the polyamine cascade by inducing the ODC mRNA level. Our results also suggest that polyamine homologs such as AP8 might be potentially useful in breast cancer therapy.

Human promyelocytic cell line HL60 has the specific binding sites for prolactin and its ornithine decarboxylase, DNA synthesis and cellular proliferation are induced by prolactin

Human prolactin (hPRL) induced ornithine decarboxylase (ODC) activity, subsequently DNA synthesis and cellular proliferation on human promyelocytic cells, HL60, cultured in a serum-free medium. HL60 cells had 2100 specific binding sites for hPRL per cell, showing a dissociation constant of 1.1 × 10 −10 M. Binding of 125I-PRL to the cells was not blocked by simultaneous addition of human growth hormone. ODC activity and DNA synthesis were activated maximally at 5 and 20 h, respectively, after the addition of 0.05 nM hPRL. These effects of PRL on cellular proliferation, ODC activity and DNA synthesis were abolished by the simultaneous addition of anti-hPRL antibody. Simultaneous addition of an irreversible inhibitor of ODC, difluoromethyl ornithine (DFMO), also abolished the inductions of ODC and DNA synthesis by hPRL. The inhibitory effect of DFMO on hPRL-induced DNA synthesis was reversed by the addition of putrescine to the culture medium. These results suggest that hPRL binds to the prolactin receptor on HL60 cells and induces ODC activity to increase cellular polyamine levels, which eventually stimulates DNA synthesis and cellular proliferation.

Effects of gastrin and difluoromethylornithine on growth of human colon cancer.

The effect of difluoromethylornithine (DFMO), a specific inhibitor of ornithine decarboxylase activity, was evaluated in vivo and in vitro on the growth of a gastrin-sensitive human colon carcinoma (WiDr). In vivo, mice bearing the tumor treated with pentagastrin had larger tumors with higher ornithine decarboxylase activity and polyamine content (P < 0.05) than mice not treated with pentagastrin. Difluoromethylornithine treatment significantly decreased ornithine decarboxylase in both the pentagastrin-treated and the untreated animals; however, DFMO had no effect on tumor volume, weight, protein, or DNA content. In cell culture, gastrin treatment increased WiDr cell number and [3H]thymidine incorporation in the presence or absence of serum. In serum-free conditions, however, gastrin stimulated cell growth without concomitantly increasing ODC activity. DFMO, on the other hand, decreased both ODC activity and growth. These studies suggest that the trophic effect of gastrin on WiDr human colon cancer is independent of ODC activity. Since gastrin treatment increased ODC activity in vivo, gastrin may interact in vitro with other factors present in serum that can alter ODC activity.

Studies on the effects of an ornithine decarboxylase inhibitor on lupus nephritis reveal a post-transcriptional modification of the enzyme.

Upregulation of ornithine decarboxylase (ODC) activity and polyamine levels is found in the kidney of MRL-lpr/lpr (lpr) mouse, an animal model of lupus. To understand the molecular genetics of ODC regulation in lpr mouse, we analyzed ODC mRNA and activity in the kidney of lpr and normal BALB/c and MRL(-)+/+ mice. Although ODC activity was significantly higher in lpr kidney, its mRNA level was lower compared to normal strains, as measured by Northern blot hybridization. Treatment of lpr mouse with difluoromethylornithine (DFMO) reduced ODC activity in lpr kidney to the level of normal strains. In contrast, ODC mRNA level increased 12-fold by DFMO treatment. These results suggest that post-transcriptional modification of ODC in lpr genetic background might be responsible for increased ODC activity and polyamines. The beneficial effect of DFMO on murine lupus suggests a pathogenic role for altered ODC regulation in lpr mouse.

The effect of difluoromethylornithine on polyamine levels in pollinated and napthaleneacetic acid-induced young tomato fruits

The polyamine synthesis inhibitor difluoromethylornithine (DFMO) inhibits growth and cell division in tomato fruits (Lycopersicon esculentum Mill. c.v. F 121) when applied after pollination by the split-stem technique. In napthaleneacetic acid (NAA)-induced fruits, however, growth is not inhibited by DFMO. We analyzed the effect of DFMO on cell division in auxin induced tomato fruits, and on the concentration of free, soluble conjugated and insoluble bound polyamines in pollinated and NAA-induced fruits, five days after pollination in the presence and absence of DFMO. Cell number was not significantly reduced by DFMO in NAA-induced fruits five days after fruit set. Free spermine, soluble conjugated and insoluble bound polyamines were higher in fruits treated with DFMO than in those treated with water. They were also higher in DFMO-treated pollinated fruits than in NAA-induced ones. Our results suggest that inhibition of cell division by polyamine synthesis inhibitor might affect further metabolism of polyamines.

α-Difluoromethylornithine decreases inhibitory transmission in hippocampal slices independently of its inhibitory effect on ornithine decar☐ylase

We tested the effect of DL-alpha-(difluoromethyl)ornithine (DFMO), a specific inhibitor of ornithine decarboxylase (ODC), on recordings in area CA1 of rat hippocampal slices. In the concentration range in which it is used as an ODC inhibitor, DFMO increased neuronal excitability and blocked paired-pulse inhibition. The effect of DFMO was reversed by perfusing the slice with normal bathing solution. These effects were not attenuated by the simultaneous addition of putrescine; thus the activity of DFMO was not related to a decrease in putrescine caused by the inhibition of ODC. Mediation by the N-methyl-D-aspartate (NMDA) receptor was ruled out because DL-2-amino-5-phosphonovalerate (APV), an NMDA antagonist, did not block the effect of DFMO. Intracellular and extracellular recordings of pharmacologically isolated IPSPs supported the notion that DFMO depressed GABAergic transmission. DFMO has frequently been used as a tool to study the role of the ODC-polyamine system in neural preparations. This report suggests that the results from such studies must be interpreted with caution. In addition, our findings raise questions about the proposed use of DFMO as a neuroprotective agent against excitotoxicity.

Effect of difluoromethylornithine (DFMO) on NSAID-induced intestinal injury in rats.

Combination therapy with difluoromethylornithine (DFMO) and a nonsteroidal antiinflammatory drug (NSAID) has been proposed for the chemoprevention of colonic neoplasia. The purpose of this study was to examine whether DFMO would affect NSAID-mediated intestinal injury. Male Sprague-Dawley rats were gavaged with 20 mg/kg of indomethacin, after seven days of exposure to drinking water with or without 2% DFMO. The rats were killed 24 or 48 hr later, and the small intestine removed for macroscopic and microscopic quantitation of intestinal injury by computerized image analysis. Seven days of DFMO alone had no effect on overall mucosal thickness, but did increase the depth of proximal intestinal crypts. Forty-eight hours after indomethacin, DFMO treatment decreased the number of indomethacin-induced ulcers and percent of the surface area ulcerated. However, DFMO also decreased the mucosal thickness, villus height, and crypt depth in indomethacin-treated rats. Thus although DFMO decreases macroscopic intestinal ulceration by indomethacin, the reduction in villus and crypt height suggests that it also impairs the mucosa's ability to recover from microscopic indomethacin-induced damage. This study shows DFMO does impact NSAID-mediated intestinal injury and therefore human trials with combinations of DFMO and NSAIDs should include monitoring for small intestinal injury.