Altered polyamine biosynthesis with aging after massive proximal small bowel resection in rat

We examined the effect of aging on polyamine biosynthesis in the small intestine. Two groups of male Wistar rats (young; 10-week-old,n=40; old; 24-month-old,n=40) underwent either a jejunal transection and reanastomosis or 90% proximal small bowel resection. The rats were sacrificed on the 1st, 2nd, 4th, and 7th postoperative day (POD). The mucosa was submitted for histological examination, weighed, and assayed for protein, DNA, RNA, and polyamine content. Ornithine decarboxylase (ODC) activity was measured and ODC mRNA in the mucosa was determined by Northern blot analysis. Compared with the values for wet weight and protein content in old rats, young rats showed significantly higher values for wet weight on the 1st and 2nd POD, and for protein content on the 1st POD, but there were no differences between young and old rats after the 4th POD. The values for ODC activity and ODC mRNA were significantly lower in old rats than in young rats on the 1st POD, but there were no differences between young and old rats after the 2nd POD. The value for putrescine in old rats was significantly lower on the 2nd POD, but was significantly higher on the 4th POD than that in young rats. The present study showed that, in old rats, the residual intestine after small bowel resection preserved sufficient adaptive capacity, but that the adaptive response was decreased. The findings in this study also suggest that a decrease in ODC mRNA expression is involved in the decreased adaptive response that occurs with aging.     

Purification of Ornithine Decarboxylase-inducing Factor from Cell-free Ascites Fluid of Ehrlich Ascites Tumor and Its Characteristics

The ornithine decarboxylase-inducing factor (ODC factor) was purified about 1,000-fold in 42% yield from the ascites fluids of an Ehrlich ascites tumor by a combination of centrifugation and concanavalin A (ConA) treatment. A single ip injection of 0.5μg of the purified factor per mouse resulted in half-maximum induction of liver ODC. The factor was found to be a trypsin- and chymotrypsin-resistant, acidic glycoprotein (pi about 4.43) with a minimum molecular weight of about 70 kilodaltons, containing a disulfide bond(s) in its functional domain. It did not react with ConA. This factor induced retrodifferentiation of liver function, causing a marked increase of prototype M2 isozyme of pyruvate kinase. It reduced liver catalase activity, and also modified thyroid hormone metabolism, reducing the serum levels of T4 and T3. These results suggest that the ODC factor is multifunctional and induces many of the changes observed in a tumor-bearing host.     

Role of peripheral polyamines in the development of inflammatory pain

Polyamines (putrescine, spermidine and spermine) are aliphatic amines that are produced by the action of ornithine decarboxylase (ODC) in a rate-limiting and protein kinase C (PKC)-regulated step. Because high levels of polyamines are found in the synovial fluid of arthritic patients, the aim of the present study was to identify the role of peripherally produced polyamines in a model of inflammatory pain induced by adjuvant. The subcutaneous injection of Complete Freund's adjuvant (CFA, 50 μL/paw) caused the development of mechanical allodynia and edema. Moreover, it increased ODC expression and activity and PKC activation. Administration of the selective ODC inhibitor DFMO (10 μmol/paw) attenuated the development of allodynia and edema and decreased ODC activity in both control and CFA-treated animals. Furthermore, administration of the PKC inhibitor GF109203X (1 nmol/paw) reduced allodynia and ODC activity in animals injected with CFA. A subcutaneous injection of putrescine (10 μmol/paw), spermidine (3–10 μmol/paw) or spermine (0.3–3 μmol/paw) into the rat paw also caused mechanical allodynia and edema. The present results suggest that endogenously synthesized polyamines are involved in the development of nociception and edema caused by an adjuvant. Moreover, polyamine production in inflammatory sites seems to be related to an increase in ODC activity stimulated by PKC activation. Thus, controlling polyamine synthesis and action could be a method of controlling inflammatory pain.     

Transport of saquinavir across human brain-microvascular endothelial cells by poly(lactide-co-glycolide) nanoparticles with surface poly-(γ-glutamic acid)

Poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) with surface poly-(γ-glutamic acid) (γ-PGA) were applied to enhance the transport of saquinavir (SQV) across the blood-brain barrier (BBB). PLGA NPs encapsulated SQV and grafted with γ-PGA to form drug carriers (γ-PGA/SQV-PLGA NPs) for crossing through a monolayer of human brain-microvascular endothelial cells (HBMECs) regulated with human astrocytes. The results revealed that a lower molecular weight of γ-PGA yielded a higher grafting efficiency of γ-PGA on PLGA NPs. In addition, γ-PGA with a low molecular weight accelerated the dissolution of SQV from γ-PGA/SQV-PLGA NPs. A higher grafting efficiency (more didecyl dimethylammonium bromide) and a lower molecular weight of γ-PGA increased the permeability of SQV across the BBB, in general. When the grafting efficiency was 85.2% at 6 kDa of γ-PGA, γ-PGA/SQV-PLGA NPs reached about 6 times the permeability of free SQV (the maximal permeability). γ-PGA could also promote the endocytosis of NPs and expression of ornithine decarboxylase by HBMECs. γ-PGA/SQV-PLGA NPs are efficacious nanoparticulate carriers in delivering antiretroviral drug across the BBB.     

Effects of late prenatal or early postnatal ethanol exposure on ornithine decarboxylase activity in brain and heart of developing rats

Pregnant rats were kept on a liquid diet containing ethanol (6.8% v/v) from the 18th day of gestation, and the developmental pattern of ornithine decarboxylase (ODC) activity was determined for the brain and heart of the offspring. In neonates exposed to ethanol throughout development, both brain and heart ODC activities increased but then declined to normal or subnormal levels; brain weights were not different from controls but heart weights were depressed. Withdrawal from ethanol at birth produced alterations in heart ODC activity similar to those in pups exposed to ethanol continuously. Neonatal withdrawal resulted in no deviations from normal ODC development in brain minus the cerebellum, but in the cerebellum, ODC levels were increased consistently. Postnatal exposure of pups to ethanol caused a decrease in heart weight and ODC activity but no significant change in brain weight or its ODC activity. These data suggest that exposure to ethanol during development produces alterations in polyamine metabolism and organ weight which are dependent upon the duration and age period of exposure.     

Effect of cold, restraint, reserpine, and splanchnicotomy on the ornithine decarboxylase activity of rat adrenal medulla and cortex

The activity of ornithine decarboxylase (EC 4.1.1.17, ODC) of the adrenal medulla and cortex was studied after subjecting rats to three different forms of stress: administration of reserpine, exposure to short periods of cold, or bodily restraint. Reserpine, cold exposure, and immobilization significantly increased ODC activity in both adrenal tissues. For medullary ODC this effect was prevented by denervation of the adrenal gland. In the cortex, splanchnicotomy reduced or prevented the stimulation of ODC activity caused by reserpine and cold exposure. When animals pretreated with reserpine were subjected to restraint, a potentiation was observed for the medullary enzyme; this effect was prevented by denervation. The cortical enzymic activity attained levels of activity similar to those observed with the individual stimuli. Exposure of rats treated with reserpine to cold led to a reduction of adrenal ODC activity, a reduction that was statistically significant only for the cortical enzyme. Denervation had no effect. Cortical ODC responses appear to be strongly influenced by hypothalamopituitary factors which, in turn, are known to be modified by stress, whereas medullary ODC activity seems to be more susceptible to changes in sympathetic activity associated with stress.     

Folate deficiency enhances arsenic effects on expression of genes involved in epidermal differentiation in transgenic K6/ODC mouse skin

Chronic arsenic exposure in humans is associated with cancers of the skin, lung, bladder and other tissues. There is evidence that folate deficiency may increase susceptibility to arsenic effects, including skin lesions. K6/ODC mice develop skin tumors when exposed to 10ppm sodium arsenite for 5 months. In the current study, K6/ODC mice maintained on either a folate deficient or folate sufficient diet were exposed to 0, 1, or 10ppm sodium arsenite in the drinking water for 30 days. Total RNA was isolated from skin samples and gene expression analyzed using Affymetrix Mouse 430 2.0 GeneChips. Data from 24 samples, with 4 mice in each of the 6 treatment groups, were RMA normalized and analyzed by two-way ANOVA using GeneSpring. Top gene ontology (GO) categories for genes responding significantly to both arsenic treatment and folate deficiency include nucleotide metabolism and cell organization and biogenesis. For many of these genes, folate deficiency magnifies the response to arsenic treatment. In particular, expression of markers of epidermal differentiation, e.g., loricrin, small proline rich proteins and involucrin, was significantly reduced by arsenic in the folate sufficient animals, and reduced further or at a lower arsenic dose in the folate deficient animals. In addition, expression of a number of epidermal cell growth/proliferation genes and cellular movement genes was altered. These results indicate that arsenic disrupts the normal balance of cell proliferation and differentiation, and that folate deficiency exacerbates these effects, consistent with the view that folate deficiency is a nutritional susceptibility factor for arsenic-induced skin tumorigenesis.     

The utility of the K6/ODC transgenic mouse as an alternative short term dermal model for carcinogenicity testing of pharmaceuticals

The use of transgenic rodents may overcome many limitations of traditional cancer studies. Regulatory perspectives continue to evolve as new models are developed and validated. The transgenic mouse, K6/ODC, develops epidermal tumors when exposed to genotoxic carcinogens. In this study, K6/ODC mice were evaluated for model fitness and health robustness in a 36-week study to determine oncogenic risk of residual DNA in vaccines from neoplastic cell substrates. K6/ODC and C57BL/6 mice were treated with T24-H-ras expression plasmid, carrier vector DNA, or saline topically or by subcutaneous injection. One group of K6/ODC mice received 7,12-dimethylbenz-[a]anthracene [DMBA] dermally. Only DMBA-treated mice developed papillomas by six weeks, increasing in incidence to 25 weeks. By week 11, many K6/ODC mice showed severe dehydration and dermal eczema. By week 32, (6/8) surviving K6/ODC mice showed loss of mobility and balance. Microscopic evaluation of tissues revealed dermal/sebaceous gland hyperplasia, follicular dystrophy, splenic atrophy, and amyloid deposition/neutrophilic infiltration within liver, heart, and spleen, in all K6/ODC mice. Pathology was not detected in C57BL/6 mice. Progressive adverse health, decreased survival, and failure to develop papillomas to the H-ras plasmid suggest that K6/ODC mice may be an inappropriate alternative model for detection of oncogenic DNA and pharmaceutical carcinogenicity testing.     

Effect of chloroform on hepatic and renal DNA synthesis and ornithine decarboxylase activity in mice and rats

Chloroform administered intraperitoneally (i.p.) to male mice and rats resulted in a dose-dependent increase in hepatic ornithine decarboxylase (ODC) activity. Maximal induction of the enzyme in mice was 10-fold and occurred at 375 mg/kg chloroform; in rats it was 52-fold and occurred at 750 mg/kg chloroform. Chloroform increased in mice and decreased in rats the rate of hepatic and renal DNA synthesis. Therefore, the induction of ODC activity in rat liver was not followed with an increase in DNA synthesis. The implications of these results to the proposed nongenetic mechanism of chloroform induction of hepatocellular carcinoma in mice and renal tumors in rats are discussed.