Premalignant alterations in rat colonic N1-acetylspermidine levels induced by 1,2-dimethylhydrazine: effects of a high corn oil dietary regimen

Recently, our laboratory has demonstrated that elevations in the levels of N1-acetylspermidine could be detected in the colonic mucosa of rats after administration of 1,2-dimethylhydrazine for 15 weeks, i.e., before the development of colon tumors. Since prior studies have indicated that diets high in fat, particularly unsaturated fat, promote the development of dimethylhydrazine-induced tumors, it was of interest to examine the effect of a corn oil dietary regimen (20% by weight) on colonic N1-acetylspermidine levels in this model of colonic adenocarcinoma. Four groups of rats were used in these studies: chow, chow + carcinogen, corn oil and corn oil + carcinogen. The carcinogen groups received weekly s.c. injections of 1,2-dimethylhydrazine (20 mg/kg body wt) for 15 weeks, while the control groups received diluent. 1 week after the last injection, animals from each group were killed, and their proximal and distal colons were resected, examined and compared with respect to polyamine levels, including N1-acetylspermidine, as well as the activities of ornithine decarboxylase, spermidine N1-acetyltransferase, and polyamine oxidase. In view of previous studies which suggested that N1-acetylspermidine levels may be elevated in the urine of patients with various malignancies, it was also of interest to examine and compare the urinary levels of this acetylated polyamine in animals from each group. The results of these experiments demonstrated that: (1) the levels of N1-acetylspermidine in the distal colonic segment were found to be increased approx. 25 and 80% in the chow + carcinogen and corn oil + carcinogen groups, respectively, compared to their control counterparts; (2) the activities of spermidine N1-acetyltransferase in the distal colonic segments of chow + carcinogen and corn oil + carcinogen animals were increased 1.5- and 2-fold, respectively, compared to control values; (3) dimethylhydrazine administration did not affect the levels of this acetylated polyamine or spermidine N1-acetyltransferase activities in the proximal colon, but, in general, did increase the levels of putrescine and spermidine as well as ornithine decarboxylase activities in both colonic segments of animals fed chow or corn oil diets; and (4) elevated urinary levels of N1-acetylspermidine did not appear to be a reliable 'premalignant' marker in this experimental model of colonic adenocarcinoma.     

1,2-Dimethylhydrazine-induced alterations in Na+-H+ exchange in rat colonic brush-border membrane vesicles

1,2-Dimethylhydrazine, in weekly subcutaneous (s.c.) doses of 20 mg/kg body weight, produces colonic tumors in virtually 100% of rodents, with a latency period of approximately 6 months. To determine whether alterations in Na+-H+ exchange existed before the development of dimethylhydrazine-induced colon cancer, rats were given s.c. injections of this agent (20 mg/kg body wt. per per week) or diluent for 5 weeks. Animals were then killed, rat colonic brush-border membrane vesicles prepared and amiloride-sensitive sodium-stimulated proton efflux was measured and compared in control and treated-preparations. The results of these studies demonstrated that dimethylhydrazine treatment: (1) significantly increased the Vmax of this exchange without altering the Km for sodium of this exchange process, utilizing the fluorescent pH-sensitive dye, acridine orange; 22Na flux experiments also demonstrated an increase in amiloride-sensitive proton-stimulated sodium influx across treated-membrane vesicles; (2) did not appear to significantly influence Na+ permeability or proton conductance in treated-preparations compared to their control counterparts; and (3) did not significantly affect the kinetic parameters of amiloride-sensitive sodium-stimulated proton efflux in renal cortex brush-border membrane vesicles using acridine orange. This data, therefore, suggests that alterations in Na+-H+ exchange in rat colonic brush-border membranes may be involved in the malignant transformation process induced by this procarcinogen in the large intestine.     

1,2-Dimethylhydrazine-induced alterations in lipid peroxidation in preneoplastic and neoplastic colonic tissues

To determine whether alterations in lipid peroxidation existed in the preneoplastic and neoplastic colonic tissues of animals treated with the procarcinogen 1,2-dimethylhydrazine, rats were injected subcutaneously with this agent (20 mg/kg body weight per week) or diluent for 5, 10, 15 and 26 weeks. At each of these time periods, animals from both groups were sacrificed, their distal colonic mucosa and/or tumors harvested, and examined and compared with respect to malondialdehyde and lipofuscin-like pigments levels. Additionally, at 26 weeks, the fatty acid composition of microsomes prepared from control, 'uninvolved' and tumor colonic tissues were analyzed and compared. The results of these experiments demonstrated that: (1) the levels of these products of lipid peroxidation were similar in the distal colons of all animals at 5 and 10 weeks; (2) at 15 weeks, however, lipid peroxidation was decreased in the distal colons of animals treated with dimethylhydrazine; (3) at 26 weeks, the levels of these products of lipid peroxidation remained lower in dimethylhydrazine-treated distal 'uninvolved' colonic mucosa and was, moreover, markedly decreased in colonic tumors; and (4) at this latter time period, differences in the fatty acid composition between tumor, 'uninvolved' and control tissues were found. These differences, however, did not appear to underlie the changes noted in the lipid peroxidation products seen in these tissues. Taken together, these findings suggest that alterations in lipid peroxidation may be involved in the colonic malignant transformation process in this experimental model.     

1,2-Dimethylhydrazine-induced alterations in colonic plasma membrane fluidity: restriction to the luminal region

Recently, work in this laboratory has shown that changes in the 'dynamic' component of fluidity, lipid composition and phospholipid methylation activity of distal colonic brush-border membranes could be detected after administration of 1,2-dimethylhydrazine to rats of the Sherman strain for 5-15 weeks, i.e., before the development of colon cancer. The present experiments were therefore conducted to: determine whether similar 'premalignant' biochemical changes could be detected in basolateral membranes of Sherman rats treated with this agent; and clarify the relationship of these membrane changes to the malignant transformation process by examining the effect of 1,2-dimethylhydrazine on these biochemical parameters in colonic antipodal plasma membranes of rats of the Lobund-Wistar strain. This particular strain of rats has previously been shown to be total resistant to the induction of tumors by 1,2-dimethylhydrazine. The results of the present experiments demonstrate that similar biochemical alterations could not be detected in the colonic plasma membranes prepared from either strain of rat treated with 1,2-dimethylhydrazine. These data support the contention that the prior biochemical membrane alterations noted in brush-border membranes of 1,2-dimethylhydrazine-treated animals are, in fact, related to the malignant transformation process and, furthermore, are confined to the luminal surface of distal colonic epithelial cells.     

1,2-Dimethylhydrazine-induced premalignant alterations in the S-adenosylmethionine/S-adenosylhomocysteine ratio and membrane lipid lateral diffusion of the rat distal colon

Prior studies by our laboratory, utilizing the 1,2-dimethylhydrazine experimental model of colonic cancer, had shown that administration of this procarcinogen for 5 weeks was found to increase phospholipid methyltransferase activity and the fluidity of rat distal colonic brush-border membranes. The present studies were conducted to further explore these 'premalignant' colonic phenomena. Male albino rats of the Sherman strain were subcutaneously injected with dimethylhydrazine (20 mg/kg body weight per week) or diluent for 5 weeks. Animals from each group were killed, distal colonic tissue harvested and the levels of S-adenosylmethionine, S-adenosylhomocysteine and decarboxylated S-adenosylmethionine measured by high performance liquid chromatography. The activity of methionine adenosyltransferase was also examined in these tissues. Additionally, brush-border membranes were isolated from the distal colonocytes of control and treated-animals and examined and compared with respect to their phospholipid methylation activities as well as their lipid fluidity as assessed by the rotational mobilities of the probes 1,6-diphenyl-1,3,5-hexatriene and DL-12-(9-anthroyl)stearic acid and translational mobility of the fluorophore pyrenedecanoic acid. The results of these studies demonstrated: (1) phospholipid methyltransferase activity in rat colonic plasma membranes was increased concomitantly with increases in the cellular levels of S-adenosylmethionine and the S-adenosylmethionine/S-adenosylhomocysteine ratio in the distal colonic segment of treated-animals; and (2) the lateral diffusion of rat distal colonic brush-border membrane lipids, as assessed by the ratio of excimer/monomer fluorescence intensities of the fluorophore pyrenedecanoate, was also increased after dimethylhydrazine administration to these animals for 5 weeks.     

Elevated N1-acetylspermidine levels in gerbil and rat brains after CNS injury

The polyamine system is very sensitive to different pathological states of the brain and is perturbed after CNS injury. The main modifications are significant increases in ornithine decarboxylase activity and an increase in tissue putrescine levels. Previously we have shown that the specific polyamine oxidase (PAO) inhibitor N1,N4-bis(2,3-butadienyl)-1,4-butanediamine (MDL 72527) reduced the tissue putrescine levels, edema, and infarct volume after transient focal cerebral ischemia in spontaneously hypertensive rats and traumatic brain injury of Sprague-Dawley rats. In the present study, N1-acetyl-spermidine accumulation was greater in injured brain regions compared with sham or contralateral regions following inhibition of PAO by MDL 72527. This indicates spermidine/spermine-N1-acetyltransferase (SSAT) activation after CNS injury. The observed increase in N1-acetylspermidine levels at 1 day after CNS trauma paralleled the decrease in putrescine levels after treatment with MDL 72527. This suggests that the increased putrescine formation at 1 day after CNS injury is mediated by the SSAT/PAO pathway, consistent with increased SSAT mRNA after transient ischemia.     

Action of spermidine, N1-acetylspermidine, and N8-acetylspermidine at apurinic sites in DNA

The cleavage efficiency of spermidine and its acetyl derivatives (N1-acetylspermidine and N8-acetylspermidine) at apurinic sites in DNA were examined by PAGE-urea analysis. The three polyamines induced different rates of cleavage when compared at 1 mM concentrations. The order of effectiveness were: spermidine greater than N8-acetylspermidine greater than N1-acetylspermidine. Thus a decrease in efficiency was observed when the first order amino-groups of spermidine were blocked. The N-8amino-group of spermidine was less effective in inducing cleavage at AP-sites than the N1-amino-group. Among several proposed models of polyamine-DNA interactions, our results can best be explained by the model postulated by Liquori et al.     

Regeneration of colonic mucosa in the rat

As part of a study of ulcer formation and healing, regeneration of colonic mucosa in rats was studied following placement of a surgical lesion. Alterations in mucosubstances and connective tissue were examined and their possible significance discussed. The sequence of events in healing was: (1) The mucosa adjacent to the lesion tipped into the lesioned area. The crypts in this mucosa became lined with cells which contained no mucus and had no striated borders. Later in the experimental period, these undifferentiated cells gave rise to cells containing carboxymucins. Cells containing sulfomucin, neutral mucin, or having striated borders arose from the carboxymucin cells. (2) An epithelial ledge of undifferentiated cells migrated onto a sulfated glycosaminoglycan, fibrous interface between necrotic and living tissue in the lesion. (3) Crypt formation began with the appearance of intraepithelial anlagen. (4) Crypts lengthened by a process of epithelial-connective tissue proliferation from the base of the crypt upwards. Following completion of connective tissue regeneration, crypts formed by invading the reestablished lamina propria. (5) The first mucous cells in the ledge contained carboxymucins. As crypt formation occurred, these cells gave rise to typical columnar absorptive cells, to cells containing sulfomucins, and to cells containing neutral mucins. (6) Lengthening of crypts ceased following the appearance of a sulfated acid glycosaminoglycan—collagenous layer deep in the submucosa.     

Thermal denaturation of mononucleosomes in the presence of spermine,spermidine, N1-acetylspermidine,N8-acetylspermidine or putrescine: implications for chromosome structure

Putrescine (a diamine) raises the thermal denaturation temperature of mononucleosomes but produces only minor changes in the overall shape of the thermal denaturation curve. This is similar to the effect of sodium ions and is consistent with nonspecific binding to the DNA of the nucleosome. At very low levels of spermidine or spermine the same simple rise in thermal denaturation temperature is seen but at higher levels (above 1 M for total spermidine concentration) the thermal denaturation curve becomes substantially sharper and the premelt region of the curve diminishes in area. The acetylspermidines display intermediate effects. The change in shape of the thermal denaturation curve was resolved into components (R1 and R2) due to mononucleosomes in their original conformation plus a component (T) induced by the presence of spermidine or spermine. The proportion of component T was substantially reduced with acetylspermidine, compared to equivalent concentrations of spermidine. Hence, we suggest that spermidine acetylationin vivo has the potential to partially destabilise the nucleosome structure, possibly in coordination with histone acetylation.     

The role of sphingomyelin synthetase and sphingomyelinase in 1,2-dimethylhydrazine-induced lipid alterations of rat colonic plasma membranes

Recently, our laboratory, utilizing the 1,2-dimethylhydrazine model of colonic adenocarcinoma, demonstrated alterations in the 'dynamic component' of fluidity in brush-border membranes prepared from distal colonocytes of rats administered this agent for 5, 10 and 15 weeks, i.e., before the development of colon cancer. Furthermore, changes in the sphingomyelin content and sphingomyelin/phosphatidylcholine molar ratio of these membranes appeared, at least partially, to be responsible for these fluidity alterations. In an attempt to elucidate the mechanism(s) involved in these dimethylhydrazine-induced lipid changes, in the present studies the activities of sphingomyelin synthetase and magnesium-dependent neutral sphingomyelinase, enzymes involved in the synthesis and degradation of this phospholipid, respectively, were examined and compared in distal colonic brush-border membranes prepared from rats after 5, 10 or 15 weeks administration of dimethylhydrazine or diluent. The results of these studies demonstrate that alterations in both these enzymatic activities can be detected after administration of dimethylhydrazine and appear to, at least in part, be responsible for the changes in membrane sphingomyelin composition noted previously. These results as well as a discussion of their possible serve as the basis for the present report.     

Formulation of N-acetylputrescine and N1-acetylspermidine in cultured human lymphocytes.

N-Acetylputrescine and N1-acetylspermidine were synthesized from putrescine in cultured human lymphocytes. They were identified by paper chromatography and t.l.c. Acetylputrescine was also identified by mass spectrometry. N-Acetylputrescine and N1-acetylspermidine were formed in untreated cells and in lectin-transformed cells.     

The formation of radiolabeled N1- and N8-acetylspermidine in various rat organs following [14C]spermidine administration

To detect the in vivo formation of acetylspermidine, three female Sprague-Dawley rats were injected intravenously with 20 muCi of [14C]spermidine. Twenty-four hours after the injection of the radiolabel, the kidneys, liver, lungs, pancreas, small intestine, spleen, stomach and thymus were removed under Halothane anesthesia. High performance liquid chromatography (HPLC) of the pooled radiolabeled extracts from each tissue demonstrated the presence of a 14C-labeled material which co-chromatographed with an acetylspermidine standard. Thin layer chromatography (TLC) of the HPLC eluent demonstrated the presence of both radiolabeled N1- and N8-acetylspermidine in the tissues. Concentrations of labeled acetylspermidine ranged from 0.27 to 1.9% of the total tissue radiolabel. The N1-to N8-acetylspermidine ratio in tissues ranged from approx. 5 to 1 in the thymus to 1.5 to 1 in the liver.     

Cell population kinetics of 1,2-dimethylhydrazine-induced colonic neoplasms and their adjacent colonic mucosa in the mouse.

The parameters of cell population kinetics of symmetrical 1,2-dimethylhydrazine-induced colonic neoplasms and their adjacent colonic mucosa in the mouse were analyzed using the fraction labeled-mitoses curve method and compared with those of three groups of epithelial cells in the crypt of the descending colon of normal mouse. The analysis of three groups of epithelial cells in the crypt of normal mouse indicates that differentiation of epithelial cells was associated not only with a smaller proliferative pool of cells but also with a shortening of the duration of G2 phase and a prolongation of mitotic time. Other parameters of cell cycle did not change significantly. The mean cell cycle time of neoplastic cells in chemically induced colonic neoplasms was similar to that of epithelial cells in normal colon, but the variance was much greater in neoplastic cells. In neoplastic cells, the proliferative pool was greater, the G1 phase prlonged, and the S phase and the mitotic time became shorter as compared to epithelial cells in normal colon. The duration of G2 phase of neoplastic cells fell between the values of presumptive stem cells and differentiating cells in normal colon, compatible with the hypothesis that neoplastic cells are transformed stem cells defective in cellular differentiation. In the colonic mucosa immediately adjacent to neoplasms, the fraction-labeled-mitoses curve showed a flat second wave, indicating that the group of cells initially labeled by the pulse became a mixture of cells, some continuing the proliferative cycle normally, some going out of cycle, some slowing down in their passage from S through G2 to M, and some being arrested in mitotic phase. Such heterogeneous behavior of cells may be closely related to expansion of neoplasms. With some assumptions, however, cell cycle parameters of those normally cycling cells were estimated: the cell cycle time and the duration of G1 phase and mitotic phase were prolonged as compared to neoplastic cells and epithelial cells of normal colon.     

Dexamethasone-induced alterations in the glycosphingolipids of rat proximal small-intestinal mucosa

Prior studies have demonstrated that glucocorticoids can influence the structure and function of several different organs, including the small intestine. However, to date, the effects of glucocorticoids on the glycosphingolipids of the rat small intestinal mucosa have not been examined. In the present experiments, male albino rats of the Sherman strain were subcutaneously administered dexamethasone (100 micrograms/100 g body wt. per day) or diluent for 4 days, and the ceramide, acidic and neutral glycosphingolipid compositions of the proximal small intestine of these animals were examined and compared. The results of these studies demonstrate that dexamethasone administration: (1) increased the content and relative percentage of hematoside (GM3) in this tissue; (2) increased the percentage of N-glycoylneuraminic acid of hematoside; (3) decreased the percentage of the long-chain base phytosphingosine of hematoside, glucosyl- and globotriaosylceramide; and (4) did not appear to influence significantly the concentration of the neutral glycosphingolipids or ceramide in this tissue. These data, therefore, indicate that dexamethasone administration induces alterations in the glycosphingolipids, particularly hematoside, of rat small-intestinal mucosa.     

Superoxide dismutase and alpha-tocopherol suppress the paraquat-induced elevation of N1-acetylspermidine and putrescine in primary culture of adult rat hepatocytes

A transient increase in N1-acetylpolyamines and putrescine (PUT) was observed in hepatocytes at the early stage of primary culture of rat hepatocytes. After pre-culture for 36 hr when the polyamine content returned to constant levels, we tested the effects of superoxide dismutase (SOD) and alpha-tocopherol on the paraquat-induced increase in N1-acetylspermidine (N1-acetyl-SPD) and PUT in the culture. Paraquat increased (N1-acetyl-SPD in a dose-dependent manner. It also increased PUT at doses between 0.1 and 1.3 mM. Both SOD and alpha-tocopherol suppressed the increase in N1-acetyl-SPD and PUT induced by paraquat. These results suggested that superoxide anion is one of the factors which increase N1-acetyl-SPD and PUT in hepatocytes. Lipopolysaccharide (LPS) little affected the polyamine concentration in the cultured hepatocytes, though it increases polyamine in mouse liver when given in vivo. These findings suggested that the formation of superoxide anion after administration of LPS in vivo is mediated by Kupffer cells.     

Distribution and changes of glycoconjugates in rat colonic mucosa during development

Summary A study was made of the modifications of glycoconjugates in rat colonic mucosa during development. Sections of the caecum, and proximal and distal portions of the colon from Sprague Dawley rats at different stages of development (embryos, fetuses, suckling, weaning and adult rats) were examined. The sections were incubated with a battery of eight fluoresceinated lectins: DBA, SBA, WGA, LFA, PNA, GS-I, UEA-I and Con A. Some sections were treated with neuraminidase, and others were submitted to sequential saponification-neuraminidase treatment prior to incubation with the lectin (WGA, PNA or LFA). The intensity of the fluorescence was evaluated and graded from absent (–) to very positive (4+). Gradual and progressive changes were seen in colonic glycoconjugates during development. These changes revealed a unique developmental pattern for each lectin, which was independent for each cellular compartment (goblet cells, luminal surface and supranuclear region). Local and regional differences, observed between the different colonic sections, were already present from early stages of development. Moreover, our study showed that for several glycoconjugates, the differentiation process in colonic mucosa began in the distal region and continued through to the proximal region, the former being the first to reach the adult pattern. In the caecum, some lectins maintained a fetal pattern throughout all the periods of development up to the adult stage.     

Aspirin suppresses 1,2-dimethylhydrazine-induced alteration of proliferative parameters in rat colonic crypts

Abstract. Human epidemiological reports and rodent experimental research data indicate a possible chemopreventive effect of regular aspirin use for decreasing risk of colon and rectum cancer incidence and mortality. We have previously demonstrated that aspirin can significantly suppress proliferative parameters in normal rat colonic epithelium when examined 24 h following an acute or chronic course of aspirin administration. To investigate whether aspirin would effectively suppress known carcinogen-induced changes in colonic epithelium, rats were given single s.c. injections of either aspirin (50 mg/kg bw) or saline on days 1–3 and either 1,2-dimethylhydrazine (DMH; 12 mg base/kg bw) or DMH vehicle on day 4 of each week for eight consecutive weeks. Rats were sacrificed 4 days after the last aspirin dose and 3 days after the last DMH or DMH vehicle dose. Using the proliferative biomarkers of proliferating cell nuclear antigen positive cells per midaxial crypt section (SCC), crypt proliferative zone height (PZ), crypt differentiated zone height (DZ), and total crypt height (CH), it was found that aspirin does suppress DMH-induced increases in SCC, PZ and CH. The findings demonstrate that aspirin has a long term (i.e. several days) protective effect against early carcinogen-induced proliferative changes in rat colonic crypts which may help account for aspirin's chemopreventive action against colon cancer.