Jejunal Crypt Cells Research Articles

Maternal dietary restriction and selenium supply alters messenger ribonucleic acid expression of angiogenic factors in maternal intestine, mammary gland, and fetal jejunal tissues during late gestation in pregnant ewe lambs1

The objectives of this study were to evaluate effects of maternal dietary restriction and Se supply on angiogenic factor mRNA expression in intestinal and mammary tissues, and jejunal crypt cell proliferation and vascularity in late-term fetal intestines. In Exp. 1, pregnant ewe lambs (n = 32; initial BW = 45.6 +/- 2.3 kg) were allotted randomly to 1 of 4 treatments. Treatments (initiated d 50 +/- 5 of gestation) were control (3.5 microg of Se.kg of BW(-1).d(-1)), Se-wheat (75 microg of Se.kg of BW(-1).d(-1)), selenate (Se3; providing 75 microg of Se.kg of BW(-1).d(-1)), selenate (Se15; providing 375 microg of Se.kg of BW(-1).d(-1)). Diets (DM basis) were similar in CP (15.5%) and ME (2.68 Mcal/kg). In Exp. 2, pregnant ewe lambs (n = 36; initial BW 53.8 +/- 1.3 kg) were allotted randomly to treatments in a 2 x 2 factorial arrangement. Factors were nutrition (control, 100% of requirements vs. restricted nutrition, 60% of controls) and dietary Se (adequate Se; 6 microg of Se.kg of BW(-1).d(-1) vs. high Se; 80 microg of Se.kg of BW(-1).d(-1)). Selenium treatments were initiated 21 d before breeding, and nutritional treatments were initiated on d 64 of gestation. Diets (DM basis) were 16% CP and 2.12 Mcal/kg of ME. In Exp. 1, Se15 increased (P = 0.07) vascular endothelial growth factor (VEGF) mRNA expression, whereas Se supplementation decreased (P = 0.06) kinase insert domain receptor (KDR) mRNA in maternal mucosal scrape on d 134 of gestation. Expression of VEGF mRNA was decreased by Se (P = 0.10) in fetal jejunum. In mammary tissue, fms-related tyrosine kinase 1 and KDR mRNA were greater in Se-wheat compared with Se3, and KDR expression was increased (P = 0.10) in Se15 compared with Se3. In Exp. 2, dietary restriction increased (P < or = 0.07) expression of mRNA for VEGF, fms-related tyrosine kinase 1, KDR, neuropilin 1, neuropilin 2, and hypoxia-inducible factor 1, alpha subunit in mucosal scrapes from maternal jejunum. In fetal jejunum, soluble guanylate cyclase, was decreased (P = 0.01) by maternal dietary restriction from d 64 to 135 of gestation. Total microvascularity in fetal jejunum was reduced (P = 0.002) by maternal dietary restriction. Mammary gland expression of VEGF, neuropilin 1, angiopoietin receptor (endothelial tyrosine kinase), and endothelial nitric oxide synthase 3 increased (P < or = 0.09), whereas angiopoietin 1 decreased (P = 0.05) due to nutrient restriction. Data indicate that expression of angiogenic factors and receptors in maternal intestine, mammary gland, and fetal jejunum are responsive to maternal nutrition and likely explain observed changes in tissue vascularity.

ErbB Activity Links the Glucagon-Like Peptide-2 Receptor to Refeeding-Induced Adaptation in the Murine Small Bowel

The small bowel mucosa is sensitive to nutrients and undergoes rapid adaptation to nutrient deprivation and refeeding through changes in apoptosis and cell proliferation, respectively. Although glucagon-like peptide-2 (GLP-2) exerts trophic effects on the gut and levels increase with refeeding, mechanisms linking GLP-2 to mucosal adaptation to refeeding remain unclear. Fasting and refeeding were studied in wild-type (WT) and Glp2r(-/-) mice and in WT mice treated with the pan ErbB inhibitor CI-1033. Experimental end points included intestinal weights, histomorphometry, gene and protein expression, and crypt cell proliferation. Fasting was associated with significant reductions in small bowel mass, decreased crypt plus villus height, and reduced crypt cell proliferation. Refeeding increased plasma levels of GLP-2, reversed small bowel atrophy, increased villus height and cell number, and stimulated jejunal crypt cell proliferation. In contrast, refeeding failed to increase small bowel weight, crypt cell proliferation, or villus cell number in Glp2r(-/-) mice. Levels of mRNA transcripts for egf, kgf, and igfr were lower in fasted Glp2r(-/-) mice. Epidermal growth factor but not insulin-like growth factor-1 restored the intestinal adaptive response to refeeding in Glp2r(-/-) mice. Furthermore, CI-1033 prevented adaptive crypt cell proliferation, Akt activation, and induction of ErbB ligand gene expression after refeeding. Up-regulation of ErbB ligand expression and intestinal Akt phosphorylation were significantly diminished in refed Glp2r(-/-) mice. These findings identify Glp2r and ErbB pathways as essential components of the signaling network regulating the adaptive mucosal response to refeeding in the mouse intestine.

Modification of gamma‐radiation response in mice by green tea polyphenols

In this study we evaluated the effect of water extracts of green tea (GT) and mixtures of green tea polyphenols (GTPs), epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC) and epicatechin (EC) on jejunal crypt survival, endogenous spleen colony formation, and apoptosis in jejunal crypt cells of mice irradiated with gamma-ray. The radioprotective effect of green tea was compared with the effect of diethyldithiocarbamate (DDC). Jejunal crypts were protected by pretreatment of GT and ECG. Administration of GT, GTPs and EC prior to irradiation resulted in an increase in the formation of endogenous spleen colonies. The frequency of apoptosis in crypt cells was also reduced by pretreatment of GT, GTPs, EGCG, ECG and EGC. In the experiment on the effect of catechins, the effects were partly contradicted in irradiated mice. The rank order of activity was ECG > EGC > EGCG > EC on intestinal crypt survival assay, EC > EGC > ECG > EGCG on the spleen colony formation assay, EGCG > EGC > EC > ECG on inhibiting the death of cells caused by apoptosis. The results indicate that GT and GTPs may have a major radioprotective effect. Each one of the catechins was a much less effective radioprotector, suggesting that total extract or a mixture of GTPs may be more effective than individual catechins.

Protein expression and distribution of murine calcium‐activated chloride channels 1 and 2 (mCLCA1/2) in epithelia of the gastrointestinal (GI) tract and kidney (Ki)

Calcium‐activated chloride channels (CLCA) have been implicated as anion channels, channel regulators and cystic fibrosis phenotype modulators, but their function remains unclear. We generated a polyclonal antibody (pAb 849) against a peptide that is common to mCLCA1/2, has ~70% aa identity with mCLCA3/4, but none with mCLCA5/6. pAb 849 detected mCLCA1 expressed in HEK293 by immunoblotting (IB) and immunoprecipitation, where 125 and 90 kDa bands suggest posttranslational modification. Flag‐tagging of mCLCA1 yielded only a 130 kDa band. pAb 849 cross‐reacted with mCLCA3 in IB with bands at 120, 100 and 75 kDa. Using immunohistochemistry and comparison with mCLCA3 (pAb p3a), mCLCA1/2 specific labelling was found in pancreas acini (apical (AP), zymogen granules (ZG)), but not in ducts. pAb 849 IB of ZG membranes yielded a 130 kDa band. Specific signals were also seen in small ducts of seromucous salivary glands (AP, intracellular (IC)) but not in excretory ducts or acini. Stomach parietal (AP, IC) and jejunal crypt cells (AP) were specifically labelled. Signals in stomach mucous and jejunal goblet cells may be due to mCLCA3 cross‐reactivity. Only AP areas of Ki distal tubules were stained. mCLCA1/2 positive tissues do not express mCLCA4, therefore the data indicate mCLCA1/2 expression in AP membranes of GI and Ki and ZG membranes, but a function as secretory proteins cannot be excluded.Funding: German CFF F04/04

Effects of level and source of dietary selenium on maternal and fetal body weight, visceral organ mass, cellularity estimates, and jejunal vascularity in pregnant ewe lambs1

Pregnant Targhee ewe lambs (n = 32; BW = 45.6 +/- 2.2 kg) were allotted randomly to 1 of 4 treatments in a completely randomized design to examine the effects of level and source of dietary Se on maternal and fetal visceral organ mass, cellularity estimates, and maternal jejunal crypt cell proliferation and vascularity. Diets contained (DM basis) either no added Se (control) or supranutritional Se from high-Se wheat at 3.0 ppm Se (SW) or from sodium selenate at 3 (S3) or 15 (S15) ppm Se. Diets were similar in CP (15.5%) and ME (2.68 Mcal/kg of DM) and were fed to meet or exceed requirements. Treatments were initiated at 50 +/- 5 d of gestation. The control, SW, S3, and S15 treatment diets provided 2.5, 75, 75, and 375 microg of Se/kg of BW, respectively. On d 134 +/- 10 of gestation, ewes were necropsied, and tissues were harvested. Contrasts, including control vs. Se treatments (SW, S3, and S15), SW vs. S3, and S3 vs. S15, were used to evaluate differences among Se levels and sources. There were no differences in ewe initial and final BW. Full viscera and liver mass (g/kg of empty BW and g/kg of maternal BW) and maternal liver protein concentration (mg/g) and content (g) were greater (P < 0.04) in Se-treated compared with control ewes. Maternal liver protein concentration was greater (P = 0.01) in SW vs. S3 ewes, and content was greater (P = 0.01) in S15 compared with S3 ewes. Maternal jejunal mucosal DNA concentration (mg/g) was greater (P = 0.08) in SW compared with S3 ewes. Total number of proliferating cells in maternal jejunal mucosa was greater (P = 0.02) in Se-fed compared with control ewes. Capillary number density within maternal jejunal tissue was greater (P = 0.08) in S3 compared with SW ewes. Selenium treatment resulted in reduced fetal heart girth (P = 0.08). Fetal kidney RNA (P = 0.04) and protein concentrations (mg/g; P = 0.03) were greater in Se-treated compared with control ewes. These results indicate that supranutritional dietary Se increases cell numbers in maternal jejunal mucosa through increased crypt cell proliferation. No indications of toxicity were observed in any of the Se treatments.

Protection by Polaprezinc Against Radiation-Induced Apoptosis in Rat Jejunal Crypt Cells

Polaprezinc, an anti-ulcer drug, is a chelate compound consisting of zinc and L-carnosine. Polaprezinc has been shown to prevent gastric mucosal injury. The anti ulcer effects of polaprezinc have been ascribed to its antioxidative property. The effect of polaprezinc on ionizing radiation-induced apoptosis was studied in the jejunal epithelial crypt cells of rats. Seven-to eight week-old Wistar rats, which were treated with 100 mg/kg of polaprezinc orally 1h before irradiation or 2% carboxymethyl cellulose sodium in controls, were exposed to whole body X-ray irradiation at 2 Gy. The number of apoptotic cells per jejunum crypt was counted in haematoxylin and eosin stained sections at 0-6 h after irradiation. TUNEL positive cells and immunopositive cells for active caspase-3 per crypt were also counted. Accumulation of p53, p21(WAF1/CIP1) and Bax expression in the jejunum after irradiation were examined by Western blot analyses. Polaprezinc treatment given prior to radiation resulted in a significant reduction in numbers of apoptotic cells, TUNEL positive cells and active caspase-3 immunopositive cells in jejunal crypt cells. Polaprezinc treatment resulted in decreases of p53 accumulation, p21(WAF1/CIP1) and Bax expression after irradiation. Polaprezinc has a protective effect against ionizing radiation induced apoptosis in rat jejunal crypt cells.

Correlation between single nucleotide polymorphisms and jejunal crypt cell apoptosis after whole body irradiation

Purpose: To identify loci concerned with radiosensitivity in a mouse model using single nucleotide polymorphism (SNP) markers.Materials and methods: We subjected 276 second filial generation (F2) mice descended from two inbred mouse strains, radiation-induced apoptosis sensitive C57BL/6JNrs (B6) and radiation-induced apoptosis resistant C3H/HeNrs (C3H), to 2.5 Gy whole-body irradiation. We quantified jejunal crypt apoptosis, performed a genome-wide survey, and identified quantitative trait loci (QTL) associated with radiation sensitivity. We expressed apoptosis levels as an apoptotic score (AS), which was equal to the number of apoptotic bodies divided by the number of crypts. We genotyped the mice for 109 SNP markers.Results: AS values were 97.7 ± 32.9 in B6 mice and 49.0 ± 24.9 in C3H mice (p < 0.01). Genome-wide analysis revealed 8 markers (2 on chromosome 9, 4 on 15, 1 on 17, and 1 on 18) affecting radiation-induced jejunal apoptosis with log odds (LOD) scores ranging from 2.11 – 3.91. We found a significant locus on chromosome 15, which was previously reported by Weil and colleagues.Conclusion: These findings support the view that the radiosensitivity of clinically normal tissue depends on variations in several genes.

In Vivo radioprotective effect of Panax ginseng C.A. Meyer and identification of active ginsenosides

This study evaluated the effect of water extracts of Panax ginseng C.A. Meyer (PG), panaxadiol (PD), panaxatriol (PT), ginsenoside Rb(1), Rb(2), Rc, Rd, Re and Rg(1) on jejunal crypt survival, endogenous spleen colony formation and apoptosis in jejunal crypt cells in gamma-irradiated mice. Jejunal crypts were protected by pretreatment with PG, Rc and Rd. Administration of PG, PD, Rd and Re prior to irradiation resulted in an increase in the formation of endogenous spleen colonies. The frequency of radiation-induced apoptosis in intestinal crypt cells was also reduced by pretreatment with PG, PD, Rb(2), Rc, Rd, Re and Rg(1). In experiments on the effects of the individual ginsenosides, the rank order of activity was Rc > Rd > Rg(1) > Rb(2) > Re > Rb(1) on intestinal crypt survival assay, Re > Rb(2) > Rd > Rg(1) > Rb(1) > Rc on the spleen colony formation assay, and Rg(1) > Re > Rd > Rc > Rb(2) > Rb(1) on inhibiting the death of cells caused by apoptosis. The results indicated that Rc, Rd and Re may have a major radioprotective effect in mice irradiated with high and low doses of radiation. When the same experiments were performed using PD and PT, it was observed that most of the inhibitory effects came from PD rather than PT.

Radioprotection in mice following oral delivery of amifostine nanoparticles

Purpose: Amifostine (Ethyol®) is an approved cytoprotective agent prescribed to reduce certain side-effects in the chemotherapy of ovarian or non-small cell lung cancer, or in radiation treatment of head-and-neck cancer. The usefulness of this drug is further hampered, because it is not effective when given orally. The objective of this part of the project was to evaluate the radioprotective efficacy of orally active amifostine nanoparticles.Materials and methods: Radioprotective efficacy was evaluated by measuring the ability of the amifostine nanoparticles (equivalent to 500 mg/Kg) to inhibit whole-body gamma irradiation -induced injury in mice. All mice received acute whole-body gamma irradiation from a Cesium-137 source and the radioprotective efficacy of the formulation was determined by measuring 30-day survival at 9 Gy, bone marrow hemopoeitic progenitor cell survival at 9 Gy and 8 Gy, and intestinal crypt cell survival at 11 Gy.Results: Thirty-day survival, hemopoietic progenitor cell survival, as well as the jejunal crypt cell survival were all significantly enhanced when the mice were treated orally with the amifostine nanoparticles 1 h prior to irradiation.Conclusions: These results clearly and unequivocally demonstrate that the amifostine nanoparticles developed in our laboratory provides significant protection from acute whole-body gamma irradiation injury in mice.

Genistein Inhibits Intestinal Cell Proliferation in Piglets

Currently 15% of U.S. infants are fed soy formulas that contain up to 14 mg of genistein equivalents/L. Our goal was to investigate the impact of dietary genistein on intestinal development. Piglets (n=8/group) were fed sow milk replacer (MR), MR+1 mg/L of genistein (LG), or MR+14 mg/L of genistein (HG) for 10 d. Formula intake, weight gain, and intestinal length and weight were similar in all groups. Average serum genistein concentration in the HG group was similar to that of soy formula-fed infants. No significant effects of genistein on enterocyte apoptosis, lactase, and sucrase activities or electrophysiologic measures were observed in jejunum or ileum. Jejunal and ileal villus heights were not significantly different, but the percentage of proliferating cell nuclear antigen-positive jejunal crypt cells in the HG was reduced 50% compared with that in MR and LG (p=0.001), indicating decreased proliferation. Enterocyte migration distance in the HG group tended to be 20% less (p=0.1) than LG or MR. Jejunal estrogen receptor beta mRNA expression in HG was half of that in LG (p=0.05), but neither was significantly different from MR. In conclusion, genistein at the level present in soy infant formula is bioactive in the small intestine and results in reduced enterocyte proliferation and migration. The lack of effect of genistein on nutrient transport and enzyme activity suggests that the impact of genistein is greater on proliferating versus differentiated intestinal cells.

Flavopiridol increases therapeutic ratio of radiotherapy by preferentially enhancing tumor radioresponse

Purpose Recently we reported that inhibition of cyclin-dependent kinases (cdks) by flavopiridol enhanced the radiation response of murine ovarian carcinoma cells in culture. The purpose of this investigation was to extend these studies to in vivo tumor models and test whether flavopiridol increases the therapeutic ratio of radiotherapy. Methods and materials Three transplantable syngeneic mouse tumors were used: mammary carcinoma (MCa-29), ovarian carcinoma (OCa-I), and a lymphoma (Ly-TH). Tumor treatment endpoints included growth delay, cure, and spontaneous lung metastases (OCa-I tumor). The normal tissue endpoint was survival of jejunal crypt cells quantified microscopically. A range of flavopiridol doses from 0.625 to 5.0 mg/kg were given systemically once or twice daily over 5, 10, or 20 days. Combined therapy flavopiridol treatments were initiated either several days before or shortly after the start of single dose or daily fractionated radiotherapy. Results The major findings of this study are that all three tumors treated with flavopiridol alone responded by tumor growth delay. Two of the tumors (MCa-29 and Ly-TH) responded in a schedule-dependent manner with larger radiation enhancement factors when flavopiridol treatment was started a few hours after irradiation (radioenhancement factors [EF] Ly-TH = 2.04, EF MCa-29 = 1.50 for single dose irradiation). When combined with fractionated irradiation (2.6 Gy daily for 10 or 20 days), flavopiridol enhanced the response of the MCa-29 tumor by a factor of 1.25–1.46. A fractional radiation dose of 6 Gy in combination with flavopiridol produced a 62.5% cure rate compared with 25% tumor cure for radiation alone. A novel finding of this study was the demonstration of antimetastatic activity of flavopiridol in addition to its effect on the local primary tumor. Both the incidence and absolute number of lung metastasis were reduced when flavopiridol followed surgical removal of the large (10 mm) primary leg tumor. The normal jejunum treated with flavopiridol and radiation responded in a schedule independent manner and the degree of radioenhancement (EF, 1.05–1.06) was much less than for any of the tumors studied. Conclusions Therapeutic gain was achieved when flavopiridol treatment was initiated either before or after the start of radiotherapy. Flavopiridol shows promising clinical potential administered alone or in combination with other cytotoxic agents, including both chemotherapy and radiotherapy.

Effect of high-selenium wheat on visceral organ mass, and intestinal cellularity and vascularity in finishing beef steers.

Twelve crossbred steers (351 +/- 24 kg initial BW) were used to determine effects of high-Se wheat on visceral tissue mass, intestinal cell growth, and intestinal cellularity and vascularity. Steers were allotted randomly by BW to one of two treatments consisting of 75% concentrate diets that supplied 1) adequate Se concentration (7 to 12 microg x kg x BW(-1) x d(-1)) or 2) high-Se concentration (60 to 70 microg x kg x BW(-1) x d(-1)). Diets were similar in composition, including 25% grass hay, 25% wheat, 39% corn, 5% desugared molasses, and 6% wheat middlings supplement on a DM basis. In the Se treatment, high-Se wheat (10 ppm Se, DM basis) was replaced with low-Se wheat (0.35 ppm Se, DM basis). Diets were formulated to be similar in CP and energy (14.0% CP, 2.12 Mcal of NEm/kg, and 1.26 Mcal NEg/kg of DM) and were offered daily (1500) to individual steers in an electronic feeding system. Diets were fed at 2.38% BW. After 126 d, steers were slaughtered, and individual visceral tissue weights determined. Concentrations of DNA, RNA, and protein of duodenum, ileum, and total small intestine were not affected (P > or = 0.33) by treatment. Similarly, RNA:DNA and protein:DNA ratios in duodenum, jejunum, ileum, and whole small intestine were not (P > or = 0.33) affected by feeding high-Se wheat. Conversely, jejunal weight was greater (P < 0.002) in steers fed high-Se wheat than in controls (916 vs. 1,427 +/- 84 g). Jejunal DNA was increased (P < 0.04) in steers fed high-Se wheat (2.95 vs. 3.56 +/- 0.19 mg/g), suggesting increased cell number. Concentrations of jejunal RNA and protein were not altered by treatment; however, because the jejunal weight increased in high-Se steers, DNA, RNA, and protein contents (grams) were greater than in control steers (P < 0.05). Vascularity of jejunal tissue decreased (P < 0.10) with high-Se wheat; however, because jejunal mass was greater for the high-Se wheat treatment, total microvascular volume was not affected by treatment. Percentage of jejunal crypt cell proliferation was not affected (P = 0.48) by treatment; however, total number of cells proliferating within the jejunum was increased in steers fed high-Se wheat. Data indicate that the lower jejunal vascularity in the diet high in Se (provided from wheat) may have resulted in increased jejunal mass to meet physiological nutrient demand. Therefore, negative effects of Se level used in this study on productive performance of feedlot steers are not expected.

Dexrazoxane's protection of jejunal crypt cells in the jejunum of C3Hf/Kam mice from doxorubicin-induced toxicity.

Dexrazoxane (DEX) is used clinically to reduce doxorubicin-induced cardiotoxicity. Because DEX inhibits anthracycline-induced toxicity, we set out to investigate DEX's ability to reduce the incidence and severity of gastrointestinal toxicity associated with anthracycline administration in C3Hf/Kam mice. Doxorubicin and idarubicin, two commonly used anthracyclines, were each examined in combination with DEX. A jejunal crypt survival assay demonstrated that DEX increased crypt survival from 40% (doxorubicin 22.5 mg/kg) to 63% at a DEX/doxorubucin dose ratio of 10:1 ( P<0.05). When doxorubicin was increased to a dose of 27.5 mg/kg, crypt survival increased from 18% to 40% at a DEX:Dox ratio of 5:1 ( P<0.05). At ratios of 10:1 and 20:1, DEX had no protective effect on idarubicin-induced crypt cell toxicity. Our findings support the use of DEX to prevent or ameliorate mucositis in patients receiving anthracycline-based therapy and the use of DEX with high-dose doxorubicin to treat refractory disease.

Stimulation of intestinal epithelial restitution by prostaglandin E(1) analogue.

5-Fluorouracil (5-FU) causes intestinal mucosal damage and malabsorption. We have recently reported that coadministration of 17 S,20-dimethyl- trans- lower right triangle (2)-prostaglandin E(1) (OP-1206), a stable synthetic analogue of prostaglandin E(1), with 5-FU to rats protects the small intestine from 5-FU-induced damage. Enterocyte proliferation would contribute to the restitution of the wounded intestinal mucosa. Thus, we investigated the effect of OP-1206 on the proliferation of rat jejunal crypt cells (IEC-6 cells) treated with 5-FU. Proliferation of IEC-6 cells was evaluated in terms of [(3)H]-thymidine incorporation and using the 3-(4,5-dimethyl-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Mucosal healing was assessed by measuring the speed of resealing across the denuded area of an IEC-6 cell monolayer. OP-1206 stimulated [(3)H]-thymidine incorporation into subconfluent IEC-6 cells pretreated with 5-FU and increased the number of IEC-6 cells. AH23848B, an EP4 prostaglandin receptor antagonist, blocked the OP-1206-stimulated [(3)H]-thymidine incorporation into IEC-6 cells. The speed of resealing across the denuded area of a wounded IEC-6 cell monolayer was found to increase following treatment with OP-1206. OP-1206 stimulated the proliferation of IEC-6 cells treated with 5-FU, indicating a possible mechanism for the protective effect of OP-1206 against 5-FU-induced damage to the small intestine. OP-1206 was shown to be active in intestinal mucosal healing.

The radioprotective effects of bu-zhong-yi-qi-tang: a prescription of traditional Chinese medicine.

We evaluated the effect of bu-zhong-yi-qi-tang, a prescription of traditional Oriental medicine, and its major ingredients on protection of the intestine and hematopoietic organs against radiation damage in this study. The jejunal crypt survival, endogenous spleen colony formation, and apoptosis in jejunal crypt cells were investigated in mice irradiated with high and low doses of gamma-rays. bu-zhong-yi-qi-tang administration before irradiation protected the jejunal crypts (p < 0.0001), increased the formation of the endogenous spleen colony (p < 0.05) and reduced the frequency of radiation-induced apoptosis (p < 0.05). In experiments on the effects of the individual ingredient of bu-zhong-yi-qi-tang, Rensan (Radix Ginseng), Danggui (Radix Angelicae gigantis), Shengma (Rhizoma Cimicifugae) and Chaihu (Radix Bupleuri) might have major radioprotective effects, and each might have different degrees of effect on these three endpoints. These results indicated that bu-zhong-yi-qi-tang might be a better agent than any one of its ingredients to satisfy all three endpoints. Although the mechanisms of this inhibitory effect remain to be elucidated, these results indicated that bu-zhong-yi-qi-tang might be a useful radioprotector, especially since it is a relatively non-toxic natural product. Further studies are needed to better characterize the protective nature of bu-zhong-yi-qi-tang extract and its ingredients.

Effects of Taxol plus radiation on the apoptotic and mitotic indices of mouse intestinal crypt cells

In this study we investigated the effect of Taxol, radiation, or Taxol plus radiation on highly proliferative normal tissue--the intestinal crypt cells of Swiss albino mice. Swiss-albino mice, 3-4 months old, were used in this study. Taxol was administered by bolus intravenously through the tail vein. Radiation was given using a linear accelerator. There were four treatment categories, which comprised a total of 34 groups. Each group consisted of five animals. The first category was a control category which comprised one group (n = 5). The second treatment category was Taxol alone which comprised three groups (n = 15). The third treatment category was radiation alone which comprised three groups (n = 15). The fourth treatment category was Taxol plus radiation which comprised 27 groups (n = 135). Mice were killed 24 h after Taxol or radiation or combined administration using ether anesthesia. Using a light microscope, apoptotic and mitotic indices were counted on jejunal crypt cells of mice that were stained with hematoxylin-eosin. Differences between groups were statistically evaluated with Student's t-test. Taxol caused a dose-dependent increase in apoptosis (P = 0.045) and decreased the mitotic index (P = 0.006) at high doses. Similarly, radiation caused a dose-dependent increase in apoptosis (P = 0.046) and decreased the mitotic index (P = 0.299) at higher radiation doses. Compared to radiation alone, Taxol caused a significant induction of apoptosis (P = 0.010). In combination, no significant radiosensitizing effect of Taxol was observed (enhancement ratio < 1), when compared to radiation alone. However, an increase in apoptosis was observed after 24 h of Taxol exposure when compared to 12 or 48 h of Taxol exposure (P = 0.0001 and P = 0.0001). These findings suggest that Taxol did not cause a radiosensitizing effect in intestinal crypt cells. However, a 24-hour pretreatment of Taxol exposure followed by radiation caused significant induction of apoptosis and reduction of the mitotic index when compared to other Taxol timing sequences. Thus, the lack of a radiosensitizing effect of Taxol in these proliferative cells may be due to enhanced mitotic death rather than apoptotic death.

The proliferative response of mouse jejunal crypt cells to radiation-induced cell depletion is not mediated exclusively by transforming growth factor alpha.

Several lines of correlative evidence link transforming growth factor alpha (Tgfa, also known as TGF-alpha) to proliferative activity in jejunal crypt cells. It is therefore tempting to hypothesize that, as a ligand of the epidermal growth factor, it mediates the compensatory proliferative burst in the crypts after radiation-induced cell killing. We have tested this hypothesis by comparing the repopulation response of wild-type and Tgfa-null mice, using the microcolony assay. Mice were exposed whole-body to (137)Cs gamma rays at a dose of approximately 1.6 Gy/min. Single doses and equal doses separated by 4 and 54 h were given. The rightward shift of the dose-response curves for 54 h was identical for wild-type and Tgfa-null mice, and there was no indication of a difference in radiosensitivity. This result indicates that Tgfa is not an essential component of the proliferative response of tissue to radiation-induced cell killing.

A Chromosome 15 Quantitative Trait Locus Controls Levels of Radiation-Induced Jejunal Crypt Cell Apoptosis in Mice

Jejunal crypt cells undergo apoptosis in response to ionizing radiation exposure. In mice the number of cells deleted by apoptosis is determined by several factors including the dose of radiation, the time of day the apoptosis level is quantified, and the strain of mouse irradiated. We previously found that the difference in radiation-induced apoptosis levels between C57BL/6J (B6) and C3Hf/Kam (C3H) mice is controlled by multiple genes, and this set of genes is distinct from that controlling thymocyte apoptosis levels in the same strain combination. Here, we report that a new quantitative trait locus on chromosome 15, Rapop5, partly accounts for the murine strain difference in susceptibility to radiation-induced jejunal crypt cell apoptosis. In addition, we show sexual dimorphism in the extent of radiation-induced jejunal crypt cell apoptosis, with female mice having higher levels.

Epinephrine stimulates the Na +-K + ATPase in isolated rat jejunal crypt cells

The effect of epinephrine on the activity of the Na +-K + ATPase was studied in isolated rat jejunal cells. The activity of the pump was assessed by measuring the ouabain inhibitable K + accumulation by the enterocytes using 86Rb as a tracer. Epinephrine stimulated significantly the Na +-K + ATPase in crypt cells but not in villus cells. This effect was still apparent in presence of propranolol and prazocin but disappeared in presence of yohimbine. Amiloride did not affect the epinephrine-induced stimulation. Calcium channel blockers and dibutyryl cAMP enhanced the activity of the pump, and exerted respectively overlapping and additive effects with epinephrine, when added simultaneously. The calcium ionophore A23187 inhibited the basal activity of the ATPase and the stimulatory effect of epinephrine disappeared in its presence. These results suggest that epinephrine stimulates the Na +-K + ATPase in jejunal crypt cells by activating α 2 receptors and decreasing intracellular calcium, and not by altering cAMP levels.

The role of the sympathetic nervous system in radiation-induced apoptosis in jejunal crypt cells of spontaneously hypertensive rats.

To evaluate the effect of the sympathetic nervous system on radiation-induced apoptosis in jejunal crypt cells, apoptosis levels were compared in spontaneously hypertensive rats (SHR), animals which are a genetic hyperfunction model of the sympathetic nervous system, and normotensive Wistar-Kyoto rats (WKY). SHR and WKY were exposed to whole body X-ray irradiation at doses from 0.5 to 2 Gy. The apoptotic index in jejunal crypt cells was significantly greater in SHR than in WKY at each time point after irradiation and at each dose. WKY and SHR were treated with reserpine to induce sympathetic dysfunction, and were subsequently exposed to irradiation. Reserpine administration to SHR or WKY resulted in a significant suppression of apoptosis. p53 accumulation was detected in the jejunum in both WKY and SHR after irradiation by Western blotting analysis. There were no significant differences in the levels of p53 accumulation in irradiated intestine between WKY and SHR. These findings suggested that hyperfunction of the sympathetic nervous system is involved in the mechanism of high susceptibility to radiation-induced apoptosis of the jejunal crypt cells.