Presurgical transarterial chemoembolization does not increase biliary stricture incidence in orthotopic liver transplant patients.

INTRODUCTION: The goal of this study was to compare the incidence of biliary strictures in orthotopic liver transplant (OLT) patients treated with previous transarterial chemoembolization (TACE) versus those with no TACE history. PATIENTS AND METHODS: A single-center retrospective review was performed on 248 patients who underwent OLT from 2006 to 2012. Patient demographic characteristics, history of TACE for treatment of hepatocellular carcinoma, OLT data, and biliary stricture data were obtained. TACE was generally performed in a segmental manner using chemotherapy to ethiodized oil mixture (1:1). Clinically significant biliary strictures resulting in cholestasis or obstructive jaundice were diagnosed by using endoscopic retrograde cholangiopancreatography. Group characteristics were compared by using the Wilcoxon rank sum test, χ(2) analysis, and Kaplan-Meier statistics with log-rank comparison. RESULTS: Forty-six patients (35 men, 11 women; median age, 58 years) with a history of pre-OLT TACE were compared with 185 patients (111 men, 74 women; median age, 54 years) with no history of TACE. TACE and non-TACE patients had 30% and 31% cumulative incidence of biliary stricture, respectively. The median time to stricture was not reached in either group. There was no statistically significant difference in biliary stricture incidence (P = .928) or time to biliary stricture development (P = .803). Biliary strictures were primarily anastomotic in location in both groups: 79% in TACE patients and 84% in non-TACE patients (P = .233). CONCLUSIONS: Selective TACE treatment of hepatocellular carcinoma in pretransplant patients does not increase the rate of posttransplant biliary strictures. These findings corroborate the safety of TACE in the treatment of hepatocellular carcinoma in potential OLT patients as a bridge to transplantation.

Acute acid exposure increases rabbit esophageal cell proliferation.

In the present study we examined whether an acute infusion of HCl into the esophagus of rabbits would cause an increase in esophageal cellular proliferation independent of morphologic evidence of cell injury. To examine this question, the distal two thirds of the rabbit esophagus was infused for 1 hour with either 40 mmol/L HCl or NSS (control), and cellular proliferation was studied 24 and 48 hours later by using bromodeoxyuridine (BrDu) to label the nuclei of dividing cells and ornithine decarboxylase (ODC) enzyme activity as a biochemical index of cell division. Although there was no gross or microscopic evidence of cell necrosis or mucosal inflammation 24 hours after H+ infusion, BrDu labeling of basal cell nuclei was significantly greater 24 hours after H+ infusion (31%+/-6%) as compared with that in control animals infused with NSS (15%+/-4%). This increase in labeling index was paralleled by a threefold greater ODC enzyme activity at 24 hours with H+ infusion. Rete pegs were infrequent in control tissues (4+/-4 rete pegs per 100 microm of esophageal length) or in animals examined 24 hours after acid exposure (4+/-2 rete pegs per 100 microm). However, rete pegs were very prominent 48 hours after acid infusion (22+/-6 rete pegs per 100 microm). A short exposure to acid can cause a significant increase in mucosal proliferation independent of injury, suggesting that esophageal cell acidification either directly or indirectly acts as a tissue mitogen.

Effect of beta-carotene supplementation on indices of colonic cell proliferation.

BACKGROUND: Epidemiologic studies have shown that consuming foods containing beta-carotene is associated with a decreased incidence of colon cancer. The validity of this association has recently been questioned. It is not known if the rate of colonic cell proliferation differs among individuals with or without a history of colonic polyps or cancer and if proliferation changes in response to beta-carotene. PURPOSE: This study was intended to (a) determine whether differences exist in colonic cell proliferation in individuals with and without prior colonic polyps or tumors, (b) demonstrate that beta-carotene accumulates in colonic mucosa following dietary supplementation, and (c) determine whether mucosal beta-carotene accumulation influences colonic cell proliferation. METHODS: Subjects were enrolled in the phase I study from June 1991 until February 1994. The participants included 20 individuals (11 males and nine females, aged 62.3 +/- 8.9 years [means +/- SD]) with normal colons (as judged by recent colonoscopy), 40 (24 males and 16 females, aged 59.6 +/- 10.1 years) with a history of colonic polyp(s), and 41 (30 males and 11 females, aged 67.2 +/- 9.7 years) with prior colon cancer. The subjects in the last two groups consumed either 30 mg of beta-carotene or placebo each morning for 3 months. This dose of beta-carotene has no known toxic effects, but it can increase the serum level by approximately 10-fold. beta-carotene concentration in serum and colonic tissue was quantitated by high-pressure liquid chromatography in samples collected before and after supplementation with beta-carotene or placebo. Cellular proliferation was assessed on the basis of tissue ornithine decarboxylase activity, urinary polyamine excretion, and proliferating cell nuclear antigen expression. The differences in colonic cell proliferation parameters due to beta-carotene supplementation, within and among different groups, were evaluated by the Wilcoxon matched-pairs signed ranked test and the Mann-Whitney test, respectively. All statistical tests were two-sided. RESULTS: Colonic cell proliferation did not differ in samples obtained from individuals with and without prior colonic polyp(s) or cancer. beta-carotene concentrations in serum and colonic tissue were significantly increased in groups receiving beta-carotene (P < .001). However, cell proliferation did not differ, as judged by any of the three measures, among samples from all experimental groups collected before and after supplementation with beta-carotene. CONCLUSIONS: Dietary supplementation with beta-carotene for a period of 3 months does not alter colonic cell proliferation in individuals with a history of colonic polyps or cancer. IMPLICATIONS: The mechanism by which beta-carotene might reduce colon cancer incidence does not appear to involve or result in a change in cell proliferation in the normal colonic mucosa as studied in individuals with a history of colonic polyps or cancer.

Urinary N1-acetylspermidine and N8-acetylspermidine excretion in normal humans and in patients with colorectal cancer.

Urinary N1-acetylspermidine (N1SPD) and N8-acetylspermidine (N8SPD) were measured in 24-hr urine specimens from 42 patients with colon adenocarcinoma and 29 healthy controls to assess their use as markers for colon cancer screening. Serial spot urines in four controls demonstrated significant fluctuations in these polyamine levels throughout the day without a distinct circadian pattern and therefore all subsequent analyses were performed on 24-hr collections. Both N1SPD and N8SPD were significantly increased in colon cancer patients compared to controls. Neither test correlated with tumor stage or location, but N8SPD was elevated in patients with poorly differentiated adenocarcinoma when compared to moderate or well-differentiated tumors. Using receiver operator characteristic (ROC) analysis, N1SPD had a higher information content than N8SPD, N1SPD + N8SPD, or the ratio of N1SPD/N8SPD and at a normal cut-off value of 4.0 nmol/mg creatinine, yielded a 95% specificity and 50% sensitivity for colon cancer.

Effects of 1,25-dihydroxyvitamin D3 on proliferation and differentiation of Caco-2 cells.

The effect of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], or calcitriol, on the proliferation and differentiation of Caco-2 cells was studied. Vitamin D receptor mRNA was detected in both pre- and postconfluent cells, and its abundance was unchanged with time and in response to calcitriol. 1,25-(OH)2D3-binding activity increased during differentiation, but there was no difference in binding between 1,25-(OH)2D3-treated and control cells. 1,25-(OH)2D3 caused a dose-dependent reduction in proliferation, as assessed by [3H]thymidine incorporation and DNA content. 1,25-(OH)2D3 significantly enhanced the normal rise in alkaline phosphatase activity during differentiation and increased alkaline phosphatase mRNA abundance. In contrast, 1,25-(OH)2D3 inhibited the normal rise in sucrase-isomaltase activity and the corresponding mRNA level, although the inhibition occurred after the initial period of cell differentiation (> 10 days postplating). Morphological analysis demonstrated that by day 12 postplating, 1,25-(OH)2D3 increased the mean dome diameter and microvillus length and density. Although 1,25-(OH)2D3 decreases the proliferation of Caco-2 cells and enhances certain parameters of differentiation, not all brush-border hydrolases respond in a similar fashion, making it necessary to interpret with caution their individual use as markers of differentiation.

Dietary calcium and vitamin D modulate 1,2-dimethylhydrazine-induced colonic carcinogenesis in the rat.

To determine whether supplemental dietary calcium and/or vitamin D deficiency are involved in modulating colon cancer induced by 1,2-dimethylhydrazine (DMH), Sprague-Dawley rats were fed diets containing either: (a) a normal content of calcium (0.87%) and phosphorus (0.60%) with 2.2 IU of vitamin D3 per g of feed (group A); (b) the same diet as group A, but with calcium and phosphorus increased to 1.80 and 0.80%, respectively (group B); or (c) a vitamin D-deficient diet with supplemental calcium (1.80%) and phosphorus (0.80%) (group C). After 6 weeks on their respective diets, one-half the animals in each group were given s.c. injections of either vehicle or DMH (20 mg/kg body weight/week) for 26 weeks. Animals were then sacrificed and the incidence of tumors as well as the number of tumors per tumor-bearing rat were determined. Colonic mucosal polyamine levels were measured after 15 weeks of exposure to vehicle or DMH, before development of histologically recognizable neoplasms. The results of these experiments demonstrated that neither calcium supplementation alone nor supplemental calcium in conjunction with vitamin D deficiency altered the incidence of colonic cancer induced by this carcinogen. Supplemental calcium, however, significantly decreased the number of rats with multiple tumors and reduced tumor size. Moreover, vitamin D deficiency abolished these protective effects of calcium on colon cancer in this experimental model. DMH treatment increased polyamine levels in the premalignant colonic mucosa in group A rats. This carcinogen-induced effect was blunted by high dietary calcium. Vitamin D-deficient, calcium-supplemented rats (group C) showed an increase in N1-acetylspermidine, but not the other polyamines, with DMH treatment.

Effect of polyamine oxidase inhibition on the colonic malignant transformation process induced by 1,2-dimethylhydrazine.

Recent studies of colon adenocarcinomas in humans and experimentally induced colonic tumors in rodents have demonstrated selective elevations in the level of N1-acetylspermidine in these malignant tissues. The exact relationship of these alterations in acetylated polyamine levels to the malignant transformation process, however, remains unclear. In order to clarify this issue, rats were given s.c. injections of 1,2-dimethylhydrazine (DMH; 20 mg/kg body wt/week) or diluent for up to 26 weeks. After 10 weeks of carcinogen treatment, one-half of the animals in each group were also concomitantly given i.p. injections of MDL 72527 (20 mg/kg body wt/week), a specific inhibitor of polyamine oxidase, until they were killed. Animals were killed after 15 weeks of DMH treatment and polyamine levels as well as the activities of polyamine oxidase, ornithine decarboxylase and spermidine-N1-acetyltransferase were measured and compared in rat proximal and distal colonic mucosa of each group. Polyamine levels were also assessed in each of these groups after 26 weeks of treatment with this carcinogen +/- MDL 72527. In addition, in view of recent studies that have indicated that polyamines may influence certain oncogenes in human colonic carcinoma cells, tumors from DMH +/- MDL 72527 were analyzed for K-ras mutations. The results of these experiments demonstrated for the first time that: (i) MDL 72527 was a specific inhibitor of polyamine oxidase in normal and malignant colonic tissue; (ii) concomitant administration of this agent with DMH enhanced the elevation of colonic N1-acetylspermidine and significantly reduced the mean colonic tumor burden, as assessed by total tumor area per rat, produced by this carcinogen alone; (iii) analysis of K-ras mutations revealed a similar incidence (62-69%) in adenocarcinomas for both groups (+/- MDL 72527); (iv) however, analysis of the K-ras-mutated and non-mutated tumors revealed that in both carcinogen-treated groups (+/- MDL 72527), tumors with such mutations were smaller than their counterparts without such genetic alterations. Moreover, MDL 72527 reduced the average size of tumors, with and without such mutations, to a similar extent.

Urinary excretion of N1-acetylspermidine and other acetylated and free polyamines in the 1,2-dimethylhydrazine model of experimental rat colon cancer.

1,2-Dimethylhydrazine (DMH) is a potent procarcinogen with selectivity for the colon. Recently, it has been demonstrated that levels of N1-acetylspermidine were elevated 2-3-fold in colonic tumors induced by this agent compared to control tissues. To determine whether alterations in the urinary levels of this acetylated polyamine or other polyamines were useful biochemical markers for colon cancer in this experimental model, rats were given s.c. injections of DMH (20 mg/kg body weight/week) or diluent for 26 weeks. One week after the last injection, control and DMH-treated animals were placed in separate metabolic cages and their urine was collected for 24 h. The urinary levels (expressed as nmol/mg creatinine) of putrescine, spermidine, spermine, N1-acetylspermidine, and N8-acetylspermidine were then analyzed by high-performance liquid chromatography. Animals from each group were then sacrificed and their colons were examined for tumors. The results of these studies demonstrated that the urinary level of N1-acetylspermidine was an excellent biochemical marker for colonic tumors induced by DMH. At 18.3 nmol/mg creatinine, N1-acetylspermidine was 100% sensitive and specific for colon cancer. Moreover, urinary levels of N1-acetylspermidine were better for this purpose than the N1-acetylspermidine/N8-acetylspermidine molar ratio, a marker previously suggested to be more specific for certain cancers than free polyamines.

1,2-Dimethylhydrazine-induced alterations in N1-acetylspermidine levels in rat distal colonic mucosa: effects of 2-difluoromethylornithine.

Recently, our laboratory has demonstrated that N1-acetylspermidine levels were increased in the distal colonic mucosa of rats administered 1,2-dimethylhydrazine for 15 and 26 weeks. In order to further explore the possible role of this acetylated polyamine in the malignant transformation process induced by this carcinogen, groups of rats were subcutaneously injected weekly with dimethylhydrazine (20 mg/kg body wt.) or diluent for 5, 10, 15 and 26 weeks +/- 1% 2-difluoromethylornithine in the drinking water. The latter agent, an irreversible inhibitor of ornithine decarboxylase, has previously been shown to inhibit colonic tumor formation in this experimental model. At each of these time periods, rats from each group were killed, their proximal and distal colonic mucosa harvested and examined, and compared with respect to polyamine levels, including N1-acetylspermidine, as well as the activities of ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine N1-acetyltransferase and polyamine oxidase. The results of these experiments demonstrated that: (1) N1-acetylspermidine levels in the proximal colonic segment of all animals were similar at each time point; (2) N1-acetylspermidine levels were also similar in the distal colons of all animals at 5 and 10 weeks. At 15 weeks, however, the level of N1-acetylspermidine was increased in the dimethylhydrazine-treated distal colonic segment secondary to increases in the activity of spermidine N1-acetyltransferase; and (3) at 26 weeks, the level of this acetylated polyamine remained higher in dimethylhydrazine-treated distal 'uninvolved' colonic mucosa and was markedly elevated in colonic tumors; (4) co-administration of difluoromethylornithine decreased the elevated levels of N1-acetylspermidine to control values in the distal colons of animals treated with carcinogen for 15 and 26 weeks; and (5) difluoromethylornithine markedly reduced the number of tumors induced by dimethylhydrazine in the distal but not proximal colonic mucosa at 26 weeks.

Differential stimulation of S-adenosylmethionine decarboxylase by difluoromethylornithine in the rat colon and small intestine.

The effects of chronic inhibition of ornithine decarboxylase (ODC) by the specific inhibitor difluoromethylornithine (DFMO) in the rat colon and small intestine on mucosal contents of polyamines, decarboxylated S-adenosylmethionine (decarboxylated AdoMet) and S-adenosylmethionine decarboxylase (AdoMet decarboxylase) activity were studied. Administration of 1% DFMO in the drinking water for 10 or 15 weeks resulted in inhibition of ODC and decreases in intracellular putrescine and spermidine contents in both proximal and distal segments of small intestine and colon. At both time points DFMO administration resulted in a dramatic stimulation of AdoMet decarboxylase activity and a rise in decarboxylated AdoMet content in the proximal and distal small-intestinal segments compared with controls, which was not seen in either colonic segment of DFMO-treated animals. This differential stimulation of AdoMet decarboxylase by DFMO in the small intestine and colon could not be entirely explained on the basis of differences in polyamine contents, which are known to regulate this enzyme activity. Kinetic and inhibition studies of AdoMet decarboxylase in control small and large intestine revealed that: (1) there was no difference in Vmax. values between the tissues; (2) the Km for AdoMet was higher in the small intestine than in the colon; and (3) the Ki for product inhibition by decarboxylated AdoMet was higher in the small intestine than in the colon. These results suggest that the differential stimulation of AdoMet decarboxylase by DFMO in the small intestine and colon may be due to different isoenzymes and could play a significant role in the regulation of polyamine contents throughout the gut.

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 N1-acetylspermidine levels and spermidine N1-acetyltransferase activity in rat colonic mucosa.

To determine whether alterations in the "reverse" or "conversion" pathway for putrescine formation were involved in the induction of colonic tumors by 1,2-dimethylhydrazine, male albino rats of the Sherman strain were given injections s.c. of this agent (20 mg/kg body weight/week) or diluent for 5, 10, 15, and/or 26 weeks. Animals were sacrificed at each of these time periods and polyamine levels, including N1- and N8-acetylspermidine, as well as the activities of ornithine decarboxylase, spermidine N1-acetyltransferase and polyamine oxidase were measured and compared in rat proximal and distal colonic mucosa of each group. The results of these studies demonstrated that: (a) N1- and N8-acetylspermidine levels were similar in the control and treated proximal colonic segments at all time periods examined; (b) N1- and N8-acetylspermidine levels were also similar in the control and treated distal colonic segments at 5 and 10 weeks; (c) at 15 weeks the level of N1-acetylspermidine, but not N8-acetylspermidine, however, was increased in the treated distal colonic segment secondary to increases in the activity of spermidine N1-acetyltransferase; and (d) at 26 weeks, the level of N1-acetylspermidine remained higher in treated distal "uninvolved" colonic tissue and were markedly elevated in colonic tumors in both segments. Based on these findings, it would appear that the reverse pathway for putrescine formation may be involved in the 1,2-dimethylhydrazine-induced malignant transformation process of the rat colon.

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.

Reversed-phase liquid-chromatographic analysis of hemodialysate from uremic patients.

With reversed-phase high-performance liquid chromatography we effected rapid and efficient separation of metabolites present in hemodialysate fluid from uremic patients on the artificial kidney. With an aqueous sodium acetate/methanol gradient as mobile phase, more than 50 ultraviolet-absorbing constituents were resolved in a 70-min chromatogram of a 100-mul sample of dialysate. Many of the metabolites could be detected in concentrations below 0.1 mg/liter. From results of ultraviolet spectrophotometric analysis and the gas- and liquid-chromatographic properties of standards, half the observed chromatographic peaks have been characterized or identified. The method has been shown to be more than 10-fold faster than comparable separations by ion-exchange techniques.