Asthma management: A new phenotype-based approach using presence of eosinophilia and allergy.

Asthma is a heterogeneous disease. The Czech Pneumology and Allergology Societies commissioned 10 experts to review the literature and create joint national guidelines for managing asthma, reflecting this heterogeneity. The aim was to develop an easy-to-use diagnostic strategy as a rational approach to the widening opportunities for the use of phenotype-targeted therapy. The guidelines were presented on websites for public comments by members of both the societies. The reviewers' comments contributed to creating the final version of the guidelines. The key hallmark of the diagnostic approach is the pragmatic concept, which assesses the presence of allergy and eosinophilia in each asthmatic patient. The guidelines define three clinically relevant asthma phenotypes: eosinophilic allergic asthma, eosinophilic nonallergic asthma and noneosinophilic nonallergic asthma. The resulting multifunctional classification describing the severity, level of control and phenotype is the starting point for a comprehensive treatment strategy. The level of control is constantly confronted with the intensity of the common stepwise pharmacotherapy, and the concurrently included phenotyping is essential for phenotype-specific therapy. The concept of the asthma approach with assessing the presence of eosinophilia and allergy provides a way for more precise diagnosis, which is a prerequisite for using widening options of personalized therapy.

Activation of the ER stress and calcium signaling in angiomyolipoma.

Renal angiomyolipomas (AMLs) are uncommon benign tumors that occur sporadically or as a part of tuberous sclerosis complex (TSC). Risk of life threatening hemorrhage is the main clinical concern. Although several evidences suggest that hyper-activation of the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway is crucial for these tumors, modulation of other metabolic pathways might affect tumor growth and progression. Therefore, we aimed to further characterize angiomyolipoma by TSC1/TSC2 expression, hypoxic status, expression of endoplasmic reticulum (ER) stress markers and calcium transport from the ER through the inositol 1,4,5-trisphosphate (IP3) receptors. Despite our expectations, angiomyolipoma were not hypoxic, as determined by absent expression of the carbonic anhydrase IX, which is a reliable marker of hypoxia. This was in accord with very low expression of TSC1 (that is associated with HIF activation) and a high expression of TSC2. Angiomyolipoma specimens also showed a significant upregulation of an anti-apoptotic marker Bcl2 when compared to healthy kidney tissue supporting the induction of pro-survival signaling. Moreover, angiomyolipoma specimens showed the overexpression of the ER stress markers XBP1, CHOP and ATF4 as well as of the mediators of calcium metabolism, namely the type 1 and 2, but not the type 3 IP3 receptors. These data suggest that the ER stress response, survival and calcium metabolism-related pathways but not hypoxia is an important component of the angiomyolipoma pathogenesis.

Divergent selection for shape of the growth curve in Japanese quail. 8. Effect of long-term selection on embryonic development and growth.

1. Changes in embryonic development and growth were analysed in relation to direct changes in postnatal growth and correlated responses in egg parameters using Japanese quail lines selected for more than 30 generations for high (HG) and low (LG) relative gain of body weight (BW) between 11 and 28 d of age, and constant BW at 49 d of age. 2. During the first 42 h as well as at the end of incubation, LG embryos were developmentally accelerated in comparison with their HG counterparts. An expected increase of line divergence across generations was observed only in traits analysed at the end of incubation. 3. In contrast to early generations, LG embryos continuously exhibited a higher BW than HG embryos and this difference temporarily disappeared only around incubation d 8. Analogous to early generations, the HG compared with LG embryos showed two periods of transient growth retardation compensated subsequently by a higher growth rate (incubation d 3-8 and 8-16). 4. More pronounced growth retardation of HG versus LG embryos in late versus early generations corresponded to more distinct decrease of HG versus LG growth rate during the first post-hatch days. Likewise, a disappearance of line BW differences on incubation d 8 characterising the late generations corresponded to the elimination of line differences in adult BW. 5. Alterations of growth pattern were associated with changes of egg size. While HG quail maintained a relatively constant adult BW and egg size across generations, the gradually increasing incidence of large eggs in the LG line allowed selection of birds with higher growth potential, which in turn amplified the line differences in the embryonic BW and eliminated the line differences in adult BW. Line differences in egg composition (larger albumen with lower density in LG compared with HG eggs) apparently contributed to the strengthening of line developmental divergence during incubation. 6. Transient lack of nutrient supply to HG embryos due to their developmental delay is probably responsible for a higher HG versus LG embryo mortality.

[Chronic obstructive pulmonary disease in the light of new guidelines -  brief summary of phenotypically oriented guidelines for nonpulmonary physicians]. / Chronická obstrukcní plicní nemoc pohledem nových doporucení -  souhrn aktuálního fenotypove zamereného standardu Ceské pneumologické a ftizeologické spolecnosti pro internisty.

INTRODUCTION: COPD is a global health and social problem. Morbidity and mortality increases in the Czech Republic. There are currently several global statements and strategies. METHODS: The Czech Pneumological and Phthisiological Society (CPFS) at the end of 2011 mandated the Section of bronchial obstruction in drafting national guidelines concerning the stable COPD. Subsequently, this document was discussed during the National Consensus Conference (COPD forum) in November 2012 and presented at series of local workshops and national conferences. National guidelines has been subject to a review and eventually posted on the website for another round of comments. DIAGNOSIS: A modern approach to COPD is a view of the patient through the pulmonary function, symptoms, exacerbation rates and the presence of specific phenotypes. CPFS identified six clinically relevant phenotypes: frequent exacerbators, COPD and asthma overlap, COPD and bronchiectasis overlap, emphysematic phenotype, bronchitic phenotype and phenotype of pulmonary cachexia. TREATMENT: TREATMENT recommendations can be divided into four elementary steps: the first step is the Elimination of all risks factors. The second one is the Standard therapy including in particular inhaled bronchodilators, pulmonary rehabilitation, and treatment of severe comorbidities. The third step is the Targeted therapy centered on clinical phenotypes of COPD. The final fourth step is the treatment of respiratory insufficiency and palliative care of the terminal COPD. CONCLUSION: The optimal treatment of COPD requires a personalized approach to the patient.

Divergent selection for shape of growth curve in Japanese quail. 7. Effect of egg storage at high temperature on embryo development and hatchability.

1. Hatching time, hatchability of fertile eggs and embryo mortality in relation to (1) physical quality of fresh eggs and (2) embryonic development during storage and incubation periods were analysed after egg storage for 1, 5 or 10 d at 30°C in the meat-type lines of Japanese quail, HG and LG, divergently selected for high and low relative weight gain between 11 and 28 d of age, respectively, and constant body weight at 49 d of age. 2. In both lines, the increase of egg storage temperature from 12°C (standard level) to 30°C increased the egg weight loss during storage, shortened the incubation period and reduced the hatching success. 3. Similar to standard egg storage temperature, LG quail hatched earlier than HG quail after egg storage at 30°C and early and late mortality of both lines increased with a prolonged period of egg storage. In contrast to the standard egg storage conditions, no line differences in hatchability were observed. 4. The results did not identify a relationship between the decrease in hatchability or embryo viability and line differences in external egg parameters as well as any important role of undesirable changes induced by a high storage temperature on albumen viscosity. 5. The pattern of embryonic death, low developmental rate of embryos and a dichotomy between the development of the extra-embryonic vascular system and the embryo itself during egg storage at high temperature implied that an insufficient nutrient supply in consequence of developmental delay could represent a key factor in increasing early and late embryo mortality.

Parthenogenetic activation of pig oocytes using pulsatile treatment with a nitric oxide donor.

The nitric oxide donor (+)-S-nitroso-N-acetylpenicillamine (SNAP) is capable of inducing parthenogenetic activation in pig oocytes matured in vitro. However, quite a long exposure to the nitric oxide donor, exceeding 10 h, is necessary for successful oocyte activation. Repeated short-term treatment with 2 mm SNAP significantly increased the activation rates despite the fact that the overall exposure time to the nitric oxide donor did not exceed 4 h. With regard to the activation rate, 12 repeated treatments lasting 10 min each were found to be the most efficient regimen (63.3%). The continuous exposure to the nitric oxide donor for the same overall time induced parthenogenetic activation in 12.5% oocytes (2-h continuous treatment with 2 mm SNAP). The development of parthenogenetic embryos increased after repeated short-term treatment with SNAP. After continuous treatment with 2 mm SNAP for 10 h, only 6.7% of the oocytes cleaved, and none developed beyond the 4-cell stage. Thirty-minute treatment repeated four times with 2 mm SNAP induced cleavage in 37.5% of the oocytes, 18.3% developed to the morula stage, and 6.7% reached the blastocyst stage. Based on the results, it is concluded that pulsatile treatment can significantly improve parthenogenetic activation rate when compared with the continuous treatment using nitric oxide donors.

Divergent selection for shape of growth curve in Japanese quail. 4. Carcase composition and thyroid hormones.

1. Changes in the relative weights of carcase, abdominal fat, breast and leg muscles, and plasma thyroid hormone concentrations occurring during the first 6 weeks of postnatal growth were analysed in males of HG and LG lines divergently selected for high and low relative body weight (BW) gain between 11 and 28 d of age, respectively, and constant adult BW. 2. The second week of postnatal life was a critical age at which the HG males exhibited a relatively faster growth in comparison to their LG counterparts and permanently exceeded LG males in the percentage by weight of carcase, breast and leg muscle. A higher production of muscle tissues was associated with lower accumulation of abdominal fat before sexual maturity. 3. In general, the plasma T(3) level of HG quail exceeded that of LG quail. Nevertheless, significant differences were found only at 14, 21 and 28 d of age, that is, in the period during which the highest inter-line differences in relative growth rate were noted. Also the T(3)/T(4) ratio followed a similar trend while plasma T(4) level showed no clear and consistent inter-line differences. 4. The results suggest that the selection for the shape of the growth curve, like the selection for body fat, modifies the carcase quality owing to shortening/prolongation of the acceleration growth phase. Individuals with a short acceleration phase of the growth curve are characterised by low carcase quality during the fattening period.

Divergent selection for shape of growth curve in Japanese quail. 3. Onset of sexual maturity and basic characteristics of early lay.

1. The onset of lay, quality of eggs during early lay and gonadal development of both sexes were analysed in meat-type lines of Japanese quail, HG and LG divergently selected for high and low relative weight gain between 11 and 28 d of age, respectively, and constant body weight (BW) at 49 d of age. 2. The LG line was sexually mature at an earlier age and lower BW than the HG line. This corresponded with the trend during embryonic and early postnatal development. 3. Analysis of gonads also confirmed earlier sexual development in the LG than in the HG line. In both lines, the growth of testes was detected about one week earlier than the growth of ovary. 4. Despite the different age and BW at onset of lay, HG and LG quail commenced lay at the same degree of maturity (about 90% of adult BW). This implied that the onset of sexual maturity could be identified as a point on the growth curve which terminates the linear phase. 5. When compared with the LG line, the HG line was characterised by a longer acceleration and shorter retardation phase of the growth curve. This difference is seen as an important determinant of line differences in growth and reproductive performance.

Quantitative analysis of embryonic kidney impairment by confocal microscopy and stereology: effect of 1,2-dibromoethane in the chick mesonephros.

1. Chick embryos in ovo were treated with a teratogenic dose of 1,2-dibromoethane (DBE) on embryonic day (ED) 3. On ED 6 and 10, histological sections of whole embryos were prepared for confocal microscopy. In parallel, mesonephroi of 10-d-old embryos were dissected for in situ staining with acridine orange (AO), a fluorescence probe for lysosomes. 2. DBE impaired differentiation of renal vessels which manifested as a delay in rearrangement of primitive renal vascular architecture on ED 6 and a significant reduction of the mesonephric vascularisation on ED 10. This was accompanied by delayed functional maturation of embryonic kidney, as suggested by staining with AO. 3. Renal vessels appeared to be more susceptible to DBE than tubules. Unequal growth of these renal components might be a cause of DBE-induced spatial disorganisation of tubular apparatus. 4. Nephrotoxic effects of DBE during the embryonic period are associated primarily with damage to the renal blood supply. 5. Confocal microscopy, stereological methods and three-dimensional reconstruction of developing tissues are useful tools to investigate pathogenic processes during embryonic development.

[The relation of GERD, bronchial asthma and the upper respiratory tract]. / Vztah refluxní choroby jícnu k astmatu bronchiale a horním cestám dýchacím.

UNLABELLED: Gastroesophageal reflux disease (GERD) is one of the most common diseases affecting upper gastrointestinal tract. It is a chronic disease, whith stadily growing incidence and prevalence in west countries during last 30 years. GERD is caused by pathologic gastroesophageal reflux (GER). GERD includes endoscopically positive, endoscopically negative and extraesophageal reflux disease. Extraesophageal symptoms of GERD have been of a growing attention and discussion during last few years. The most discussed topics are the relation of GERD and bronchial asthma (BA), chronic cough and symptomatology from ear, nose and throught (ENT) regions, but also non - cardial chest pain and many others. AIM: In our clinic we ran a 5 years study which aim was to evaluate the presence of GERD in patients with bronchial asthma, chronic cough and affections from ENT regions. To assess if 3 months GERD treatment would improve lung function, subjective complaints (cough) and GERD control in asthmatics; if this treatment would allow to step - down with antiasthma medication. To assess if 3 months GERD treatment can improve objective and subjective assessments in patients with chronic cough and findings in ENT regions. As for GERD, we evaluated the improvement of pH and subjective complaints (pyrosis). METHODS: We examined 86 patients with different severity of bronchial asthma, 54 patients with chronic cough and 31 patients with ENT symptoms. All patients underwent 24 hour esophageal pH metry, spirometry with lung function evaluation and objective ENT examination by flexible laryngoscopy. In case of pathologic finding on 24 hour pH-metry 3 months full antireflux treatment with proton pump inhibitors (PPI) and prokinetics was introduced. After 3 months of GERD treatment we performed control 24 hour esophageal pH metry, control spirometry and ENT examination by flexible laryngoscopy. Patients were asked to make their subjective symptoms assessments. RESULTS: We found that GERD prevalence in patients with respiratory symptoms was very high. Three months GERD treatment improved lung function (FEV1) with statistical significance (p = 0.0319), and so improved GERD control (in 60.7% of patients with high statistical significance p = 0.0009). Subjective complaints (cough) also improved in most patients. 3 months GERD treatment did not allow to step down with maintenance BA therapy according to GINA guidelines, but it enabled to decrease the rescue medications in 50% of patients. Patients with chronic cough can benefit from GERD treatment as cough improved in 75.8% of patients. CONCLUSION: Objective findings as well as subjective complaints improved in 75% of patients with ENT symptomatology. GERD control (DeMeester score and pyrosis if present) was highly statistically significant in all three groups of patients. It is necessary to mention, that there is a high presence of nocturnal acid breakthrough (NAB) in patients with respiratory symptoms: 30.3 % in patients with bronchial asthma, 63.6 % in patients with chronic cough and 45 % of patients with ENT manifestations.

Divergent selection for shape of growth curve in Japanese quail. 2. Embryonic development and growth.

1. Embryonic growth and development were analysed using meat type lines of Japanese quail, HG and LG, divergently selected for shape of the growth curve. A total of 1020 embryos of generations 9, 10 or 13 were used for analysis. 2. Considerable inter-line differences were observed in the rate of embryonic development. When compared to HG, LG embryos appeared to be developmentally accelerated during the first 42 h of incubation (larger blastoderm diameter, more somites and higher frequency of more advanced Hamburger-Hamilton stages) as well as at the end of the prenatal period (more embryos with the yolk sac inside the body cavity, shorter incubation period). This corresponded with the trend in postnatal development. 3. Embryonic growth of both lines exhibited an exponential trend. However, considerable inter-line differences were noted in the rate of embryonic growth. Initial growth retardation compensated subsequently by a higher growth rate of HG vs LG quail, characterised the lines after hatching. The same growth pattern repeated three times during the prenatal period (between d 0 and 3, 3 and 8, and 8 and 16). 4. The repeated occurrence of transient decreases in growth rate of the developmentally delayed HG line could be associated with a delayed onset of genetically determined physiological functions mediating utilisation of nutrient supply. 5. Hence, different shapes of growth curves in two genotypes with similar growth potential reveal inter-line differences in physiological age persisting during the whole ontogenesis.

Assessment of nephrotoxicity in the chick embryo: effects of cisplatin and 1,2-dibromoethane.

Morphological symptoms of mesonephric kidney damage were analysed in chick embryos treated with nephrotoxic agents--CDDP or DBE. The drugs were administered intraamniotically on ED 3 at doses 0.03 and 0.3 microg CDDP or 100 and 300 microg DBE per embryo. Body weight and absolute and relative measures of the mesonephroi (length, weight and form) were evaluated on ED 10. The higher doses of both agents affected the mass of this organ significantly. Simultaneously, a dose-dependent increase of renal malformations was detected in treated embryos, while the incidence of gross and cardiovascular defects was low (DBE) or absent (CDDP). Together with less pronounced effects on the total body growth, the results gave evidence for a higher sensitivity of the mesonephros to toxic insult when compared to the whole organism. A direct cytotoxic effect multiplied by concomitant injury of blood supply seemed to be the main cause of CDDP nephrotoxicity. In the case of DBE, damage to the mesonephros was probably associated with a primary impairment of the vascular network. The chick embryo in ovo provides a promising system for the assessment of nephrotoxic effects induced by prospective therapeutic agents and environmental contaminants during the prenatal period.

Effects of 1,2-dibromoethane on haematopoiesis in the chick embryo.

1. Chick embryo in ovo was used to investigate the effects of 1,2-dibromoethane (DBE) on haematopoiesis at a developmental stage where the primitive erythroid cells divide and differentiate in circulation. 2. Early after DBE treatment on embryonic day 3, annexin V/propidium iodide labelling showed acute cell death of erythroid elements, which was subsequently compensated for by the release of immature cells into the circulation. Simultaneously, the comet assay indicated increased DNA damage in DBE-exposed blood cells when compared with controls. 3. After embryonic day 5, there was no indication for ongoing prominent cell death in the DBE-treated group. However, the DNA damage assessed by the comet assay persisted until embryonic day 10 in the peripheral blood cells, and for even longer in cells from thymus and bursa. 4. The kinetics of DNA fragmentation in both erythroid and lymphoid cells implied genotoxic damage by DBE to the stem cells of the definitive elements and transmission of this damage through the successive cell generations. 5. The early chick embryo provides a suitable alternative to mammalian models for investigation of long-term effects of xenobiotics on haematopoiesis.

[Pathologic gastroesophageal reflux in patients with bronchial asthma]. / Patologický gastroezofageální reflux u pacientu s astmatem bronchiale.

Gastrooesophageal reflux (GER) and asthma bronchiale are frequent diseases. Asthma affects some 3-10% of adults. Gastrooesophageal reflux is present in some 45-89% asthmatic patients. Symptoms of GER are not only gastrooesophageal, and recently increased attention is focused on extraoesophageal symptoms where in particular the relationship of GER and asthma or chronic cough is investigated. At our clinic we implemented a pilot study with the objective to monitor the presence of pathological GER in patients with asthma and to assess whether antireflux therapy will influence the respiratory complaints of the patients. The group was formed by 14 patients selected at random with different severity of asthma and different symptoms of GER. The patients had a baseline examination evaluating the presence of GER (24-hour pH metry) and pulmonary function (FEV1). In case of a pathological GER the patients were treated by antireflux therapy and then check-up examinations were made. It was found that after treatment of GER in patients with asthma in particular subjective symptoms improved such as cough and pyrosis which leads to a substantial improvement of the quality of life. On the other hand reflux treatment did not exert a basic effect on pulmonary functions and it was not possible to reduce the medication of asthma.

[The comet assay: principles of the method and clinical utilization]. / Kometový test: princip metody a klinické aplikace.

BACKGROUND: Single cell gel electrophoresis or comet assay is used at present in the world to study of DNA damage, DNA repair and apoptosis. The aim of the work is to introduce the principle of comet assay to the medical community and to give a basic survey of its possible clinical applications. The article includes our first experience with this method in detection of apoptosis in bone marrow cells of patients with myelodysplastic syndrome (MDS). METHODS AND RESULTS: The whole bone marrow aspirates from 6 patients with MDS and 7 control persons were processed by alkaline version of comet assay and the degree of DNA fragmentation in individual cells was quantified using Image Analysis System. In comparison with controls, the patients with diagnosis RA and RARS exhibited in bone marrow significantly elevated number of cells with high level of DNA breaks, reflecting most probably the apoptotic cleavage of DNA. In contrast, the patient in proliferative stage of the disease (MDS-CMML-->AML) exhibited decreased frequency of apoptotic cells, well below the control level. CONCLUSION: Our results correspond with the data published on the occurrence of apoptosis in particular types of MDS. Comet assay represents a simple and cheap technique, applicable in clinical hematology to specify the diagnosis, to monitor the disease progress and efficacy of therapy not only in patients with MDS but also in other diseases resulting from an imbalance between proliferation and apoptosis.

Embryotoxicity of 1,2-dibromoethane in chick embryos in ovo: early and late effects.

Embryotoxic effects of 1,2-dibromoethane (DBE), a compound still widely used in industry, have been analyzed using chick embryos in ovo. Administration on embryonic days (ED) 3,4 or 5 induced dose-dependent embryotoxicity, manifested namely as the early embryonic death. A serious disturbance of the vascular system represented probably the main cause of strong embryolethality and growth retardation in the group of survivors. Amniotic bands in the parietal region and defects of brain and aorta prevailed in the malformation spectrum registered on ED 10. The local character of early induced changes suggests a direct effect of DBE itself in the embryotoxic action. This process is probably accomplished through interaction with lipids in cell membranes owing to the hydrophobic character of DBE molecules. The results, however, did not exclude an involvement of reactive metabolites in final embryotoxicity via the formation of DNA-adducts. In any case, a decreasing embryotoxicity of DBE with the age of treated embryos documented that the onset of liver function, assumed to occur on ED 5, did not increase the efficacy of DBE bioactivation. Our results confirmed the short-term embryotoxic properties of DBE reported in rat embryonic cultures. In addition, the in ovo system enabled us to reveal also long-term consequences represented namely by the formation of amniotic bands, not detectable in studies in vitro. The results obtained with the chick embryo in ovo confirmed the suitability of this system for embryotoxicity testing.

The role of cell death in limb development of rats manifesting Lx allele on different genetic backgrounds.

Control of vertebrate digital pattern is a phylogenetically old mechanism. Animal strains with abnormal digital counts are a useful model system to study tissue, cell and molecular factors involved in limb patterning. The aim of this study was to investigate rat limb morphogenesis on gestation days 13 to 16 in normodactylous, polydactylous and oligodactylous fetuses where the deviation from the normal pentadactylous phenotype is caused by interaction of mutant Lx allele with different genetic backgrounds. General development was assessed by measurements of crown-rump length, and limb morphogenesis by hand and foot plate width. Skeletogenesis was studied histologically and by whole mount staining with Alcian Blue and Acridine Orange. Cell death was demonstrated by supravital staining and fluorescence microscopy and by standard histology on serial sections. No phenotypic differences among the groups were noted on day 13. On day 14, the oligodactylous hind limb buds were more spiky than normal and had well-developed preaxial necrotic site (foyer preaxial primaire) which was normally observed only on day 15. This area of programmed cell death was severely attenuated in polydactylous limb buds. Pollex triphalangy manifested as increased hand plate width from day 15. Also hind limb buds width differed by this stage between groups. No acceleration or retardation of skeletogenesis was observed in abnormal limbs. The data confirm the crucial role of spatial and temporal patterns of morphogenetic programmed cell death in control of digital pattern.

Role of kidney S9 in the mutagenic properties of 1,2-dibromoethane.

The mutagenic properties of 1,2-dibromoethane (DBE) were studied in the Ames Salmonella typhimurium assay using the strains TA 1535 and TA 100. Kidney S9 fraction alone did not modify the direct mutagenic activity of DBE; but an addition of kidney S9 to liver S9 fraction yielded a higher mutagenic activity of DBE than with liver S9 fraction alone. Moreover, the addition of glutathione (GSH) to kidney S9 increased the mutagenic activity of DBE. Methimazole, a competitive inhibitor of the flavin-containing monooxygenase, reduced mutagenic activity suggesting that this enzyme may contribute to renal damage from DBE. No mutagens could be detected in the urine of rats treated with DBE.

Genotoxic and embryotoxic effects of 5-bromodeoxyuridine in the chick embryo.

Chick embryos were exposed intra-amniotically to the thymidine analog bromodeoxyuridine (BrdU) in order to study its embryotoxic and genotoxic effects. Teratogenic effects were observed at doses of BrdU which failed to produce mitotic inhibition, clastogenic effects or any significant increase in sister chromatid exchanges. Clastogenic effects and depressed cell proliferation were observed only at high embryolethal doses. Thus, BrdU-induced teratogenicity was independent of genotoxic effects manifested at chromosomal level. On the contrary, a significant increase of DNA single strand breaks was detected even 24 hours after the administration of teratogenic dose. BrdU incorporation in the DNA does not appear to prevent embryonic cells from mitotic proliferation. Whether the single strand breaks in DNA would ultimately lead to BrdU-induced teratogenesis in chick embryos remained undetermined.

Mutagenic and teratogenic effects of cyclophosphamide on the chick embryo: chromosomal aberrations and cell proliferation in affected and unaffected tissues.

Chromosomal aberrations and cell proliferation were analyzed in the chick embryo blood, limb bud, and facial tissues 12 and 24 hours after cyclophosphamide (CP) administration on day 3. The cytogenic findings were compared with teratogenic effects evaluated on incubation day 8. Low dose (0.3 micrograms) resulting in heart defects exclusively, increased the frequency of aberrant cells with simultaneous depression of cell proliferation in blood only. High dose of CP (6 micrograms), besides the heart defects, also induced facial clefts and limb malformations, and strong clastogenic effects associated with mitotic inhibition were observed in all tissues investigated. The results support the idea that the consequences of mutagenic action of cyclophosphamide--cell cycle delay and excessive death of cells with unstable aberrations--result in abnormal morphogenesis.