Iron deficiency depresses cell proliferation in hamster cheek pouch epithelium

Iron deficiency anaemia was induced in hamsters by feeding a low iron diet coupled with weekly bleeding. To assess cell proliferation, the stathmokinetic agent vinblastine sulphate was administered and cell birth rates were calculated from cumulative mitotic indices. The rate was significantly reduced in epithelium from iron-deficient animals. The uptake of tritiated thymidine [( 3H]TdR) was also significantly reduced in these animals. Results of both stathmokinetic and labelling experiments indicate that cell production in the cheek pouch epithelium of iron-deficient animals is impaired.     

Cell proliferation in dimethylhydrazine-induced colonic adenocarcinomata following cytotoxic drug treatment.

A stathmokinetic technique was used to study cell proliferation in dimethylhydrazine-induced adenocarcinomata of rat colon following treatment with cytotoxic drugs. The rate of cell division was significantly increased three days after treatment with 5,7-dihydroxytryptamine and seven days after treatment with 5-fluorouracil. Acceleration of tumour cell proliferation following 5,7-dihydroxytryptamine treatment was inhibited by treating animals with the antiseritoninergic drug Xylamidine Tosylate. Acceleration of tumour cell proliferation following 5-fluorouracil treatment was inhibited by treating animals either with the antiseritoninergic drug BW501 or with the histamine H2-receptor blocking drug Cimetidine.     

Cell population kinetic profile of the mouse thymus, and the changes induced by prednisolone.

The cell population kinetic parameters of the thymus in BALB/c mice have been estimated using stathmokinetic and [3H]TdR techniques in both control animals and animals treated with prednisolone. FLM data were analysed by computer using the Gilbert program. The study showed that prednisolone had an inhibitory effect mainly in the DNA synthesis phase and in G1. Stathmokinetic data also showed a decrease in the cell birth rate and an increase in the apparent cell cycle time (or potential doubling time) after treatment. The labelling index, the mitotic index and the growth fraction were also decreased. The study also shows a good agreement between the data obtained by stathmokinetic and [3H]TdR techniques.     

Circadian variation in the mitotic rate of the rat corneal epithelium. Cell divisions and migration are analyzed by a mathematical model

A stathmokinetic method was used to study the diurnal variation in the mitotic rate (MR) of the rat corneal epithelium, and in the adjacent conjunctival epithelium. A prominent circadian variation in cell proliferation was observed in both epithelia, both showing almost the same pattern, which may indicate that both tissues are submitted to the same regulatory mechanisms. The average rate of cell renewal during a 24 h period indicated a mean cell renewal time of 12.3 days. This is longer than previously assumed. The MR declined toward the central cornea. Based on the above observations and the known centripetal migration of cells in the corneal epithelium, we have developed a mathematical model showing isomorphism with the renewal of the corneal epithelium.     

Iron Deficiency Alters Discrete Proteins in Rat Caudate Nucleus and Nucleus Accumbens

Young rats (21 days old) made nutritionally iron deficient, by feeding them a semisynthetic diet containing skimmed milk for 5 weeks, had significantly lowered hemoglobin levels (5.2 +/- 4 g/100 ml). The nonheme iron content in caudate nucleus was decreased by 47%. The behavioral response of iron-deficient rats to apomorphine (2 mg/kg) and the density of 3,4-dihydroxyphenylethylamine (dopamine) D2 receptors, as measured by [3H]spiperone binding in caudate nucleus, were significantly reduced by 70 and 53%, respectively. The possibility that nutritional iron deficiency may affect protein content in brain was investigated by measuring the apparent concentration of proteins in caudate nucleus and nucleus accumbens from iron-deficient and control animals using two-dimensional gel electrophoresis. The data indicate that iron deficiency can affect content in these two brain regions. Significant changes in the content of 10 proteins were noted in the caudate nucleus and nucleus accumbens in iron-deficient rats. The albumin level was significantly increased in both regions studied, whereas the neuron-specific enolase level was increased in the nucleus accumbens and the glial fibrillary acidic protein level was reduced in the caudate nucleus. The significance of these protein content changes, as well as a reduction in content of a 94-kilodalton protein (a molecular size similar to that of the D2 dopamine receptor), remains to be established.     

CELL POPULATION KINETIC PROFILE OF THE MOUSE THYMUS, AND THE CHANGES INDUCED BY PREDNISOLONE

The cell population kinetic parameters of the thymus in BALB/c mice have been estimated using stathmokinetic and [³H]TdR techniques in both control animals and animals treated with prednisolone. FLM data were analysed by computer using the Gilbert program. The study showed that prednisolone had an inhibitory effect mainly in the DNA synthesis phase and in G₁. Stathmokinetic data also showed a decrease in the cell birth rate and an increase in the apparent cell cycle time (or potential doubling time) after treatment. The labelling index, the mitotic index and the growth fraction were also decreased. The study also shows a good agreement between the data obtained by stathmokinetic and [³H]TdR techniques.     

The metaphase-arrest technique applied to human cervical epithelium. I. Technique and dose-response studies

In order to study cell proliferation in intact human cervical epithelium, a technique involving metaphase arrest has been utilized. Metaphase accumulation is observed following intraepithelial administration of vincristine sulphate, at a predetermined optimum dose of 50 micrograms/ml. A significant delay before the onset of stathmokinetic activity is demonstrated; thereafter linearity of accumulation is apparent over a 4-hr period following injection. The technique appears applicable to the estimation and comparison of cell production rates in vivo of normal cervical epithelium, cervical intraepithelial neoplasia, wart-affected cervical epithelium and early invasive carcinoma. However, individual values are likely to be imprecise in isolation, indicating the need to study relatively large numbers of subjects in each group. Some practical difficulties are discussed.     

The age distribution of cells in stratified squamous epithelium

In structured tissues such as stratified squamous epithelium the age distribution of cycling cells in a particular part of the tissue is influenced by cell migration. Age distributions are derived for the proliferative cells of stratified squamous epithelium, dependent on the mode of migration; and the effect of using analytical methods which assume exponential or rectangular age distributions is discussed with particular reference to the analysis of stathmokinetic experiments.     

The Metaphase-Arrest Technique Applied to Human Cervical Epithelium I.

In order to study cell proliferation in intact human cervical epithelium, a technique involving metaphase arrest has been utilized. Metaphase accumulation is observed following intraepithelial administration of vincristine sulphate, at a predetermined optimum dose of 50 μg/ml. A significant delay before the onset of stathmokinetic activity is demonstrated; thereafter linearity of accumulation is apparent over a 4-hr period following injection. The technique appears applicable to the estimation and comparison of cell production rates in vivo of normal cervical epithelium, cervical intraepithelial neoplasia, wart-affected cervical epithelium and early invasive carcinoma. However, individual values are likely to be imprecise in isolation, indicating the need to study relatively large numbers of subjects in each group. Some practical difficulties are discussed.     

Analysis of intestinal cell proliferation after guanethidine-induced sympathectomy. II. Percentage labelled mitoses studies

Neonatal administration of guanethidine-sulfate results in an alteration of the cell proliferative pattern of the small intestinal epithelium of the young adult rat. Sympathectomy with guanethidine has previously been shown to depress mitotic, labelling, and total cellular migration indices while increasing the generation cycle time (Tc) of small intestinal crypt cells as measured by a stathmokinetic method. The present study showed that the G1, S and G2 phases of the crypt cell cycle are altered by sympathectomy, G1 accounting for most of the increase in Tc. In addition, the percentage of [3H]-thymidine labelled crypt cells is reduced and the duration of crypt cell transit is lengthened by guanethidine-induced sympathectomy.     

Effects of iron deficiency and chronic iron overloading on mitochondrial heme biosynthetic enzymes in rat liver

The effects of iron deficiency and iron overloading on the mitochondrial enzymes involved in heme synthesis were studied in rat livers. The in vitro activities of several of the enzymes in this pathway were differentially influenced by the in vivo iron status of the animals. delta-Aminolevulinic acid synthase was slightly increased in iron-overloaded animals, but remained normal in iron-deficient animals (0.58 +/- 0.09, 0.91 +/- 0.19 and 0.61 +/- 0.12 nmol delta-aminolevulinic acid/mg per h). Copro- and protoporphyrinogen oxidase activities were increased (20 and 60% above controls) in iron-deficient animals. In contrast, coproporphyrinogen oxidase was decreased by 20%, while protoporphyrinogen oxidase remained unchanged in iron-overloaded rats. These variations of activities were not due to changes in the affinity of these enzymes toward their substrates, as coporphyrinogen had the same Km in each case (0.62 +/- 0.05 M) as did protoporphyrinogen (0.22 +/- 0.035 M). Thus, the Km did not vary with the treatment received by the animals. Ferrochelatase activity was measured by both the pyridine hemochromogen method and by measurement of zinc protoporphyrin with endogenous zinc as substrate. In all cases, ferrochelatase was found to be able to synthesize zinc protoporphyrin with endogenous zinc as substrate. However, the apparent Km of zinc chelatase for protoporphyrin was significantly different in the three groups of animals with Km,appProto, app = 2.4 +/- 0.1 10(-7), 4 +/- 0.3 10(-7) and 9.10 +/- 0.05 10(-7) M in iron-overloaded, control and iron-deficient animals, respectively. When ferrochelatase activity was measured by pyridine hemochromogen, identical results were observed in iron-deficient and control animals but decreased by 45% in iron-overloaded animals. The mitochondrial heme content was also decreased by 40% in iron-overloaded rats but unchanged in either iron-deficient or control rats.     

BM-derived cells restore expression of peroxiredoxin V in the airways following acute naphthalene injury in mice

BACKGROUND: Naphthalene-induced respiratory tract toxicity in mice is characterized by specific and rapid loss of the Clara cell population, which is restored only after several days. The sources of restoration of this cell population remain unclear. We investigated whether BM-derived cells participated in the process of epithelial restoration following naphthalene toxicity compared with bacterial infection. We further investigated the role of BM-derived cells in restoration of expression of peroxiredoxin V (PRXV), one of the major proteins of antioxidant defense, specifically expressed in the bronchial epithelium. METHODS: We transplanted GFP-tagged BM cells into 5 Gy-irradiated C57BL/6 recipients. Following 1 month of recovery, experimental animals were subjected to 250 mg/kg naphthalene i.p. An additional group of animals received intratracheal instillation of Escherichia coli to induce acute bacterial inflammation. Animals were killed at 1-12 days after naphthalene and analyzed immunohistochemically. RESULTS: Recipients' cells of bronchial epithelium demonstrated significantly reduced levels of PRXV expression following naphthalene. In animals with acute bacterial inflammation, PRXV levels were not reduced in epithelium and participation of BM-derived cells in epithelial restoration was minimal. Following naphthalene, GFP(+) cells were present in large numbers in lung parenchyma and epithelium of conducting airways starting at 1 day following injury. GFP(+) progeny of BM cells was the major source of PRXV in the epithelium. DISCUSSION: These data suggest that BM-derived cells may provide a source of antioxidant protection of airways by expression of PRXV in a model of acute epithelial respiratory tract toxicity.     

Iron metabolism in Trypanosoma lewisi infection: serum iron and serum iron-binding capacity.

Progressive changes in iron levels, total iron binding capacity and hematocrit values in sera of rats infected with Trypanosoma lewisi are described. The host dietary group were: (1) complete or full complement; (2) iron-deficient, and (3) pair-fed or calorically restricted. The hematocrit values of T. lewisi-infected rats given the various diets were not significantly different from those of the controls. The decrease in total iron binding capacity (TIBC) of rats inoculated with T. lewisi and fed complete and pair-fed diets ranged up to 15% over uninfected controls. TIBC levels in rats fed an iron-deficient diet and inoculated with T. lewisi ranged up to 32% over uninfected controls. TIBC levels of deficient infected rats were significantly different from the controls from day 90 to infection to the end of the observation period. Serum iron (SI) values of non-infected rats regardless of dietary regimen showed significantly higher values than T. lewisi-infected animals between days 95 and 120. The average SI value, for this period, in adequately fed control rats was 204 +/- 7 microgram/100 ml as compared to 172 +/- 5 microgram/100 for trypanosome-infected rats. SI levels of rats on a pair-fed diet and infected with T. lewisi decreased to 17% over uninfected controls. SI levels of animals on an iron-deficient diet and infected with T. lewisi decreased up to 76% over uninfected controls.     

Helicobacter pylori perturbs iron trafficking in the epithelium to grow on the cell surface

Helicobacter pylori (Hp) injects the CagA effector protein into host epithelial cells and induces growth factor-like signaling, perturbs cell-cell junctions, and alters host cell polarity. This enables Hp to grow as microcolonies adhered to the host cell surface even in conditions that do not support growth of free-swimming bacteria. We hypothesized that CagA alters host cell physiology to allow Hp to obtain specific nutrients from or across the epithelial barrier. Using a polarized epithelium model system, we find that isogenic ΔcagA mutants are defective in cell surface microcolony formation, but exogenous addition of iron to the apical medium partially rescues this defect, suggesting that one of CagA's effects on host cells is to facilitate iron acquisition from the host. Hp adhered to the apical epithelial surface increase basolateral uptake of transferrin and induce its transcytosis in a CagA-dependent manner. Both CagA and VacA contribute to the perturbation of transferrin recycling, since VacA is involved in apical mislocalization of the transferrin receptor to sites of bacterial attachment. To determine if the transferrin recycling pathway is involved in Hp colonization of the cell surface, we silenced transferrin receptor expression during infection. This resulted in a reduced ability of Hp to colonize the polarized epithelium. To test whether CagA is important in promoting iron acquisition in vivo, we compared colonization of Hp in iron-replete vs. iron-deficient Mongolian gerbils. While wild type Hp and ΔcagA mutants colonized iron-replete gerbils at similar levels, ΔcagA mutants are markedly impaired in colonizing iron-deficient gerbils. Our study indicates that CagA and VacA act in concert to usurp the polarized process of host cell iron uptake, allowing Hp to use the cell surface as a replicative niche.     

ANALYSIS OF INTESTINAL CELL PROLIFERATION AFTER GUANETHIDINE-INDUCED SYMPATHECTOMY

Neonatal administration of guanethidine-sulfate results in an alteration of the cell proliferative pattern of the small intestinal epithelium of the young adult rat. Sympathectomy with guanethidine has previously been shown to depress mitotic, labelling, and total cellular migration indices while increasing the generation cycle time (T_C) of small intestinal crypt cells as measured by a stathmokinetic method. The present study showed that the G₁, S and G₂ phases of the crypt cell cycle are altered by sympathectomy, G₁ accounting for most of the increase in T_C. In addition, the percentage of [³H]-thymidine labelled crypt cells is reduced and the duration of crypt cell transit is lengthened by guanethidine-induced sympathectomy.     

Melatonin-induced suppression of the pinealectomy-stimulated rat adrenal cortex mitotic activity

Pineal involvement in the regulation of adrenocortical mitotic activity has recently been suggested. It has been shown that melatonin (Mel) decreased the mean mitotic activity rate (MMAR) of the adrenal cortex both in vivo and in organ culture. The goal of the present study was to test the influence of pinealectomy (PX) and/or Mel-treatment on the MMAR of adrenocortical cells, as well as on the adrenal weight in rats. The stathmokinetic method was used in the study. It was found that PX significantly increased the MMAR of the adrenocortical cells. Moreover, Mel suppressed the proliferogenic effect of PX on the rat adrenocortical cells. Melatonin alone did not significantly affect the mitotic activity of the adrenal cortex. None of the three experimental procedures, i.e. Mel, PX and Mel-treatment of pinealectomized animals significantly affected the adrenal weight. The present data suggest that Mel may be involved in the inhibitory control of adrenocortical cell proliferation.     

Epidermal chalones and squamous cell carcinomas. The growth inhibitory effects of aqueous epidermal extracts (G1 and G2 chalones) on the epidermis and on a transplantable keratinizing carcinoman in nude mice.

Balb/c/nu nude mice that had been transplanted with a moderately differentiated squamous cell carcinoma were injected i.p. with different doses of epidermal chalone, and control animals were injected with saline. The labelling indices (H3TdR) and the mitotic rate (stathmokinetic method with vinblastine sulphate) were determined. In the untreated animals, both the labelling index and the mitotic rate of the tumor were considerably higher than in the epidermis, and the rate of cell birth was almost twice that of the epidermis. Higher doses of chalone were needed to reduce the labelling index for the tumour than for the epidermis, and there was generally a less pronounced dose/response relationship in the tumours than in the epidermis. The same was true of the mitotic rate but here the results were not as obvious as for the labelling index. A possible explanation of the results may be that the tumour cells are less sensitive than epidermal cells to the injected chalones, or that reduced vascularization of the transplanted tumour may lead to reduced access of chalone, or that tumour necrosis may pay a role. However, it is evident that the tumour cells react less than the epidermis to both the G1 and the G2 chalone, and thus the findings of this study do not provide any evidence against the theory that epidermoid transplanted tumours are less sensitive to epidermal chalones than normal tissue of the same histogenetic origin.     

Nonparametric analysis of stathmokinesis

The nonparametric analysis of the stathmokinetic experiment presented in this paper is an extension of procedures by Jagers and Staudte. The method allows one to estimate, under very general assumptions, the first two moments of the residence time in successive cell cycle phases. Approximate formulae for the mean square errors of the estimates are derived. Applications include experimental stathmokinetic data for various cell lines, both analyzed and not analyzed previously. Comparison proves that the nonparametric method is very accurate whenever it can be applied. Results of analysis of the stathmokinetic data are also discussed from the viewpoint of the variability of the cell cycle generation time.     

VARIATION IN THE DURATION OF MITOSIS IN THE CRYPTS OF LIEBERKUHN OF THE RAT; A CYTOKINETIC STUDY USING VINCRISTINE

The variation in the duration of mitosis ( t _m) with cell position in the small intestinal crypts of the adult rat has been measured by a stathmokinetic technique using vincristine. The value for the whole crypt column was 0.43 hr, or 26 min. At the bottom of the crypt t _m was in excess of 1 hr, but rapidly decreased and throughout the greater part of the proliferative compartment was between 0.40 and 0.50 hr. At the top of the proliferative compartment an increase in t _m was demonstrated.
If the value of 0.43 hr for the whole crypt column is correct, then one argument for postulating the formation of metabolic DNA during differentiation in the small bowel epithelium of the rat becomes invalid. Variations in t _m within the crypt have been shown to increase the values of cell velocity obtained from cumulative birth rate diagrams. Finally further evidence has been presented for the existence of a slowly dividing subpopulation of cells at the base of the crypt. These cells may be important in crypt repopulation after damage with phase specific anti-tumour drugs.