Changes in erythropoietic activity of the rat plasma in the course of posttransfusion polycythaemia

The aim of this study was to determine the erythropoietic activity of the plasma of polycythaemic rats and of one of its protein fractions playing a role in erythropoiesis regulation. The erythropoietin activity and that of the erythropoiesis inhibitor varied in the examined plasma samples at definite time periods after induction of polycythaemia. It was demonstrated that the most suitable time of plasma collection for the inhibitor investigation is the period between 115 and 187 h after the first transfusion, and that in some cases separation of this factor from erythropoietin present simultaneously in the plasma is indispensable in order to reveal the inhibitory activity. The erythropoiesis inhibitor administered jointly with erythropoietin was found to exert no influence on erythropoiesis either in normal or in polycythaemic recipients of the tested plasma.     

The effect of various stage maturity of erythrocytes upon proliferatio rate of erythroid cells in "crown" of erythroblastic islands

Joint cultivation of small amounts of young and mature erythrocytes with bone marrow erythroblastic islands during 24 hours in liquid culture system results in erythropoiesis stimulation due to increase of number of cells in the state of mitosis in island's "crown". Prolonged joint cultivation and/or joint cultivation with plenty of premature and mature erythrocytes leads to inhibition of the erythropoiesis in the state of mitosis in the island's "crown". The maximum of the inhibitory effect was shown by erythrocytes from polycythemia rats.     

Investigations on erythropoiesis in newborn rats. Dependence of erythropoiesis in newborn rats on the intensity of the haemopoietic processes in the lactating mothers.

The intensity of haemopoietic processes was investigated in 7, 9, 11, 14 and 19-day-old suckling rats in relation to the intensity of these processes in their mothers. The rate of the haemopoietic processes in newborn rats was determined on the basis of 59Fe incorporation into the blood and haemopoietic organs. The activity of the erythropoietic system in lactating rat females was stimulated by haemorrhage and inhibited by erythrocyte transfusion. Anaemization of lactating rats by haemorrhage did not stimulate erythropoiesis in the suckling rats. Posttransfusion polycythaemia in the lactating mothers inhibited erythropoiesis in the suckling rats beginning with the 9th day of life. This phenomenon became more pronounced with the age of the rats.     

Intermittent hypoxia in patients with unexplained polycythaemia.

The aetiology of polycythaemia is unclear in up to 30% of patients. Twenty patients with unexplained polycythaemia were investigated to see whether they had an intermittent hypoxic stimulus to erythropoiesis that was undetected by conventional investigations for hypoxic secondary polycythaemia. Overnight polygraphic sleep studies showed that five patients had prolonged nocturnal hypoxaemia. Their arterial oxygen saturation was below 92%, the level at which appreciable hypoxic stimulation of erythropoiesis occurs, for 26-68% of the time for which they were studied. Considerable evidence is accumulating that intermittent hypoxia is a potent stimulus to erythropoiesis, and clinicians should consider the possibility of nocturnal hypoxia in patients with unexplained polycythaemia. Appropriate investigation will lead to the correct diagnosis of polycythaemia secondary to hypoxia in some cases previously regarded as idiopathic, and treatment may then be planned accordingly.     

Dynamics of hematological blood parameters of white rats in postnatal ontogenesis

In experiments on Wistar rats, dynamics of blood hematologic and rheological parameters was studied from birth to the 3-month age. Results of this study indicate an intensive activity of the red bone marrow anlagen for the first 3 weeks of postnatal ontogenesis. This is manifested as an increase of the number of cells in the erythron system, a change of the erythrocyte acidic resistance, and a shift in the leukocytic formula. The boundary between the 2nd and 3rd weeks of ontogenesis is a critical period: there occur a sharp deceleration of erythropoiesis and a change of the age-related erythrocyte composition and of the blood leukocyte ratio towards the parameters characteristic of adult rats. The rise in the number of leukocytes and erythrocytes does not stop after this period. Until the 4-week age, the deformability index increases to be accompanied by an increase of hemoglobin hydration and improvement of the erythrocyte toroid shape. Subsequently, inversion of these processes occurs.     

Interactive inhibition of erythroid 59Fe utilization by benzene metabolites in female mice

Using radioiron uptake into erythrocytes as a measure of hematopoiesis, it was demonstrated that benzene inhibited bone marrow function in female mice. Hydroquinone was marginally effective, but the inhibition occurred only at the highest dose tested (100 mg/kg). The combination of phenol and hydroquinone was more effective in reducing erythrocyte production than either chemical given alone. Catechol given alone was not inhibitory but when phenol was added to catechol, erythropoiesis was suppressed, as observed for the phenol and hydroquinone combination. It appears that benzene toxicity may be the result of cooperative inhibitory effects produced by its metabolites.     

Intestinal absorption in the mechanically obstructed rat intestine: Protection by prostaglandins

Intestinal obstruction inhibits amino acid absorption. The inhibition, being dependent on the pathological changes of the absorptive epithelium, was considered as an index of injury and measured after varying periods of obstruction and after pretreatment with clindamycin, indomethacin, 16, 16-dimethyl-PGE2 or arachidonic acid. A reduction in amino acid uptake was apparent after 2h of obstruction and was increasingly evident after 4, 6 and 18h. During the late phase (after 6h), inhibition was partly prevented by pretreatment with clindamycin, but the antibiotic was ineffective during the early phase (within the first 2h). Bacterial colony counts of luminal contents of rats obstructed for 2h, were not different from counts obtained in controls, but significantly lower than counts in rats that have been obstructed for 6h. Pretreatment of rats with 16,16-dimethyl-PEG2 or with arachidonic acid prevented the early inhibitory effects of the obstruction. The findings suggest that the early inhibition in amino acid uptake may be related to metabolic changes that are correctable by the administration of 16,16-dimethyl-PGE2 or of arachidonic acid. The inhibition, during the late phase, is mainly related to an overgrowth of the enteric bacteria.     

Effect of estrogen on induction of micronuclei by mutagens in male mice

The effect of estrogen on the induction of micronucleated polychromatic erythrocytes (MPCE) by mutagens was examined in male mice. In the dose-response study, a dose-related inhibition of the mitomycin C (MMC)-induced MPCE frequency by estradiol (E2) treatment was observed. In the time study, inhibitory effects of E2 on MPCE frequency by MMC were observed when MMC was administered at 0 or 1 day after injection of E2. The most effective protocol for inhibition was when E2 and MMC were used on the same day. Of mutagens other than MMC, only vincristine (VCR) showed a significant decrease in MPCE frequency when used together with E2. Benzo[a]pyrene (BaP) and 5-fluorouracil (5-FU) showed no significant decrease in MPCE frequency. The data suggest that the induction of micronuclei by mutagens is inhibited by treatment with estrogen, and this could result in a sex difference in the sensitivity of mice employed in the micronucleus test. Mechanisms of the inhibitory effects of estrogen are discussed; these might include a suppression of erythropoiesis and a possible effect on the cell membrane permeability of erythroblasts.     

Intestinal absorption in the mechanically obstructed rat intestine: protection by prostaglandins

Intestinal obstruction inhibits amino acid absorption. The inhibition, being dependent on the pathological changes of the absorptive epithelium, was considered as an index of injury and measured after varying periods of obstruction and after pretreatment with clindamycin, indomethacin, 16,16-dimethyl-PGE2 or arachidonic acid. A reduction in amino acid uptake was apparent after 2h of obstruction and was increasingly evident after 4, 6 and 18 h. During the late phase (after 6 h), inhibition was partly prevented by pretreatment with clindamycin, but the antibiotic was ineffective during the early phase (within the first 2 h). Bacterial colony counts of luminal contents of rats obstructed for 2 h, were not different from counts obtained in controls, but significantly lower than counts in rats that have been obstructed for 6 h. Pretreatment of rats with 16,16-dimethyl-PGE2 or with arachidonic acid prevented the early inhibitory effects of the obstruction. The findings suggest that the early inhibition in amino acid uptake may be related to metabolic changes that are correctable by the administration of 16,16-dimethyl-PGE2 or of arachidonic acid. The inhibition, during the late phase, is mainly related to an overgrowth of the enteric bacteria.     

Metrifonate and dichlorvos: Effects of a single oral administration on cholinesterase activity in rat brain and blood

Cholinesterase activities in rat forebrain, erythrocytes, and plasma were assessed after a single oral administration of metrifonate or dichlorvos. In 3-month-old rats, the dichlorvos (10 mg/kg p.o.)-induced inhibition of cholinesterase reached its peak in brain after 15–45 min and after 10–30 min in erythrocytes and plasma. Cholinesterase activity recovered rapidly after the peak of inhibition, but did not reach control values in brain and erythrocytes within 24 h after drug administration. The recovery of plasma cholinesterase activity, in contrast, was already complete 12 h after dichlorvos treatment. Metrifonate (100 mg/kg p.o.) had qualitatively similar inhibition kinetics as dichlorvos, albeit with a slightly delayed onset. Peak values were attained 45–60 min (brain) and 20–45 min (blood), after drug administration. Apparently complete recovery of cholinesterase activity was noted in both tissues 24 h after treatment. The dose-dependence of drug-induced inhibition of cholinesterase in rat blood and brain was determined at the time of maximal inhibition, i.e., 30 min after dichlorvos treatment and 45 min after metrifonate treatment. The oral ED₅₀ values obtained for dichlorvos were 8 mg/kg for brain and 6 mg/kg for both erythrocyte and plasma cholinesterase. The corresponding oral ED₅₀ values for metrifonate were 10 to 15 times higher, i.e., 90 mg/kg in brain and 80 mg/kg in erythrocytes and plasma. In rats deprived of food for 18 h before drug treatment, the corresponding ED₅₀ values for metrifonate were 60 and 45 mg/kg, respectively, indicating an about two-fold higher sensitivity of fasted rats to metrifonate-induced cholinesterase inhibition compared to non-fasted rats. Compared to 3-month-old rats, 19-month-old rats showed a higher sensitivity towards metrifonate and dichlorvos. At the time of maximal inhibition, there was a strong correlation between the degree of cholinesterase inhibition in brain and blood. These results demonstrate that single oral administration of metrifonate and dichlorvos induces an inhibition of blood and brain cholinesterase in the conscious rat in a dose-dependent and apparently fully reversible manner. While the efficiency of a given dose of inhibitor may vary with the satiety status or age of the animal, the extent of brain ChE inhibition can be estimated from the level of blood ChE activity.     

Effect of erythrocytes from intact, polycythemic, and anemic rats on erythropoiesis in erythroblastic island culture

Comparison of influence of erythrocytes of different stages of maturity, and their amount on erythropoiesis in erythroblastic island culture revealed their dose-dependent and maturity-dependent inhibiting effect on erythropoiesis. Erythrocytes of polycythemic rats inhibit maturing and formation of erythroblastic islands more intensively than erythrocytes of normal on anemic rats.     

Effect of Trypanosoma brucei brucei on erythropoiesis in infected rats

Anemia generated from African trypanosome infection is considered an important symptom in humans and in domestic animals. In order to recover from anemia, the process of erythropoiesis is essential. Erythropoiesis is affected by erythropoietin (EPO), an erythropoietic hormone, supplying iron and inflammatory and proinflammatory cytokines. However, the role of these factors in erythropoiesis during African trypanosome infection remains unclear. In the present study, we analyze how erythropoiesis is altered in anemic Trypanosoma brucei brucei (interleukin-tat 1.4 strain [ILS])-infected rats. We report that the packed cell volume (PCV) of blood from ILS-infected rats was significantly lower 4 days after infection, whereas the number of reticulocytes, as an index of erythropoiesis, did not increase. The level of EPO mRNA in ILS-infected rats did not increase from the third day to the sixth day after infection, the same time that the PCV decreased. Kidney cells of uninfected rats cultured with ILS trypanosome strain for 8 hr in vitro decreased EPO mRNA levels. Treatment of both ILS and cobalt chloride mimicked hypoxia, which restrained the EPO-production-promoting effect of the cobalt. Messenger RNA levels of β-globin and transferrin receptor, as markers of erythropoiesis in the bone marrow, also decreased in ILS-infected rats. Levels of hepcidin mRNA, which controls the supply of iron to the marrow in liver, were increased in ILS-infected rats; however, the concentration of serum iron did not change. Furthermore, mRNA levels of interleukin-12, interferon-γ, tumor necrosis factor-α, and macrophage migration inhibitory factor in the spleen, factors that have the potential to restrain erythropoiesis in bone marrow, were elevated in the ILS-infected rats. These results suggest that ILS infection in rats affect erythropoiesis, which responds by decreasing EPO production and restraining its function in the bone marrow.     

The switch from larval to adult globin gene expression in Xenopus laevis is mediated by erythroid cells from distinct compartments

The transition of hemoglobins during metamorphosis of Xenopus laevis involves replacement of the larval erythrocytes by adult ones, suggesting that the developmental control of this event depends upon the growth characteristics of the precursor cells. To identify the erythroid precursor cells and to investigate their developmental fate, we analyzed the distribution of stage-specific globin mRNAs by northern blotting in dorsal and ventral fragments of stage 32 embryos after in vitro culture as well as presumptive erythropoietic tissues of tadpoles during metamorphosis. The histological analysis shows that erythrocytes differentiate only in ventral fragments, suggesting that the ventral blood islands and most likely also the dorsolateral mesoderm are the primary sites of erythropoiesis. We also demonstrate that the first generations of erythrocytes, already express the predominating larval-specific alpha-globin mRNAs. The globin mRNA patterns obtained from presumptive erythropoietic tissues suggest an important role of circulating precursor cells in larval erythropoiesis, whereas the liver appears to be the main site of formation and maturation of the adult erythrocytes. Tentatively we propose that anuran erythropoiesis is dependent upon a self-perpetuating stem-cell line and that the larval and the adult erythrocytes are derived from successive generations of erythroid precursors, whose commitment may be imposed by the erythropoietic sites.     

Morphofunctional properties of red blood cells in humans during seven-day “Dry” immersion

Healthy male volunteers were subjected to seven-day “dry” immersion. After that, morphological and biochemical features of erythrocytes, erythropoiesis intensity, including the indices of iron metabolism and erythropoietin, lipid and phospholipid spectrum of the plasma membrane of erythrocytes, and the efficiency of binding and release of oxygen by hemoglobin were studied. The studies were performed before immersion, at the last seventh day of immersion, and on the 7th and 15th days of the recovery period. We found that seven-day “dry” immersion tended to change morphological composition of red blood, erythropoiesis intensity, and metabolic indices in erythrocytes. Seven-day simulated microgravity resulted in significant changes in the indices of oxygen-transporting function of erythrocytes, probably, due to changes at the membrane level and, particularly, in phospholipid fractions. These changes have no clinical importance, because all of them returned to the baseline after the 15-day recovery period. Substantial variability of data is related to an individual response of the body to stress induced by experimental conditions.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxic and cytotoxic effects of the antiviral drug, ribavirin, was studied in rat bone marrow by employing the micronucleus assay. Ribavirin in doses of 10, 15, 20, 30, 50, 75, 100 and 200 mg/kg, and cyclophosphamide (CP) 40 mg/kg (only for sex-difference study) were injected intraperitoneally. Bone marrow was collected at 24 h and 48 h following the injection. To evaluate the recovery, the bone marrow was also sampled at 72 h from 20, 100 and 200 mg/kg treated rats. The micronucleus assay was conducted according to the standard procedure. Ribavirin elevated the incidence of micronuclei (except 10 mg/kg) in erythrocytes (P<0.01). The micronucleated polychromatic erythrocytes showed the initial steep increase at 15 and 20 mg/kg dose level, then with the gradual increase, possibly due to the limited metabolism and action of higher doses. The incidence of micronucleated normochromatic erythrocytes was not dose dependent. The effect was more at 48 h than 24 h due to prolonged toxicity of the drug or its metabolites, and by 72 h, recovery was observed eventhough the genotoxicity was significant. The PCE% decreased as the dose was increased up to 75 mg/kg, then without much difference between two higher doses. Only 100 mg/kg ribavirin and CP showed more toxicity on male rats. Cytotoxicity was seen due to hindered erythropoiesis or cell destruction. Our findings suggest that ribavirin is genotoxic and cytotoxic agent for rat bone marrow.     

Characteristics of physico-chemical properties of circulating erythrocytes in the post-anoxia period

The normobarometric hypoxic hypoxia stimulates different alterations in the fundamental parameters of physicochemical properties of the peripheric blood erythrocytes and has a stimulating effect on the system of the erythropoiesis in post-hypoxia period.     

THE EFFECT OF POLYCYTHAEMIC SERUM ON THE PROLIFERATION OF RAT BONE MARROW CELLS IN VITRO

The effect of experimental polycythaemia on the rate of proliferation of erythrocytic precursor cells was investigated by means of an in vitro technique. The serum obtained from polycythaemic rats was found to inhibit significantly ³H-thymidine incorporation in normal rat bone marrow cells in vitro, as compared with normal serum. Autoradiographic analysis revealed that this inhibition resulted from a reduction in the number of labelled bone marrow cells. The inhibition proved to be specific to the erythrocyte precursor cells; the labelling index was reduced in the erythrocytic cell population by 21–50% (P < 0.001) at different incubation times, while the effect on the granulocytic cell population was negligible. It is deduced that an inhibitor substance responsible for the effects observed is present in polycythaemic serum. It is proposed that this factor is the ‘erythrocytic chalone'. The results support the general view that triggering of stem cells is not the only mode of regulation of erythropoiesis, but that the rate of proliferation of the precursor cells in the erythron is also regulated.     

A possible dual mechanism involved in the inhibitory influence of suckling on luteinizing hormone release in the ovariectomized rat.

A study was conducted to further understand involvement of the endogenous opioid peptides in suckling-induced inhibition of LH release in ovariectomized rats. The first experiment was designed to determine the effect of an opioid antagonist, naloxone (NAL, 1.0 mg. kg-1h-1), on the increase in peripheral LH concentration 18 h after pup removal and on the decrease in LH concentration 18 h after pup return. Infusion of NAL during the 18 h after pup removal or during the 18 h after pup return neither accentuated nor attenuated serum LH concentrations. The second experiment was designed to determine the effect of NAL on peripheral LH concentrations in continuously suckled rats. Serum LH increased (p less than 0.10 and p less than 0.005, respectively) in response to 18 and 36 h of NAL infusion. The third experiment was designed to determine the effect of pup removal during NAL infusion on serum LH. Peripheral LH concentrations were not different in the rats treated with 36 h of NAL infusion whether they were suckled for the duration of the infusion or nonsuckled for the last 18 h of infusion. These results suggest that suckling may inhibit LH release through two mechanisms. The first may be an opioid-independent or enhanced opioid tone mechanism important for the initiation of the inhibitory effect of suckling on LH release, while the second may be an opioid-dependent mechanism important for the sustained inhibitory effect of suckling on LH release.     

Influence of polycythaemia on erythrodieresis

Influence of the polycythaemia on peritoneal and splenic macrophage function and NO production was studied. Interaction of peritoneal and splenic macrophages with autologous erythrocytes was examined. Concentration of nitrate ions (stable metabolites ofnitric oxide) was measured in peritoneal lavage, splenic cells suspension, blood, as well as in supernatant of peritoneal and splenic macrophages. The data obtained suggest that splenic macrophages from intact rats are more active than the peritoneal ones. Splenic macrophage activity decreases whereas that of peritoneal macrophages stays unaltered in polycythaemia. Concentration of nitrate ions is significantly increased in this condition.