Epicatechin exerts dual action to shield sickling and hydroxyurea-induced myelosuppression: Implication in sickle cell anemia management.

Hydroxyurea (HU) is the key drug to treat Sickle cell anemia (SCA). However, its treatment is associated with the liability of myelosuppression. The present study aimed to investigate the potential of epicatechin as a supplementation therapy for the symptomatic management of SCA under HU therapy. A panel of experiments were performed at first to observe epicatechin's effect on sickling and hemolytic behaviour using SCA patient's blood (ex vivo). Thereafter, the effect of HU in the presence or absence of epicatechin was investigated on cytokine inhibition in rat splenocytes (ex vivo) as well as alterations in hematological parameters and kidney function tests in rats (in vivo). Then, any effect of epicatechin on pharmacokinetic modulation of HU in rats was elucidated along with the underlying mechanism using a battery of in vitro and in vivo models. Epicatechin exhibited potent action on anti-sickling, polymerization inhibition, and erythrocyte membrane stability. It did not show any inherent hemolytic activity and reduced TNF-α level during concomitant administration with HU. Based on hematological changes in rats, epicatechin treatment aided to the beneficial effect of HU and prevented the treatment-linked disadvantageous effects of HU like neutropenia. The plasma exposure of HU was significantly augmented in rats upon simultaneous oral administration of epicatechin with HU. Down-regulation of Oatp1b2 and catalase possibly contributed to the pharmacokinetic interaction of HU. Epicatechin is found to be a promising candidate and should be explored at a reduced dose level of HU towards offsetting the dose-dependent myelosuppressive effect of HU under the frame of supplementation therapy in SCA.

Attenuation potentials of royal jelly against hydroxyurea-induced infertility through inhibiting oxidation and release of pro-inflammatory cytokines in male rats.

Hydroxyurea (HDU), a class of antineoplastic drugs, has a powerful efficacy in the treatment of several types of malignancies. However, it has multiple adverse effects including reduced fertility, especially in males. Thus, 60 male albino rats were used to investigate the chemoprotective potentials of royal jelly on HDU-induced testicular damage. Animals were gastro-gavaged with HDU (225 or 450 mg kg-1 bw day-1) before royal jelly (100 mg kg-1 bw day-1) for 60 days. Blood samples and testicles were collected, and spermatozoon was obtained. In a dose-dependent manner, the sperm count, motility and liveability, and testosterone, GSH, and catalase concentrations were decreased in HDU groups, whereas MDA, FSH, LH, IL-6, and IFN-γ expression levels were increased. Germinal epithelium degeneration, germ cell sloughing, reduction in the number of luminal spermatozoa, interstitial congestion, and severe leukocyte infiltration besides no glandular secretion in most of the acini were identified. However, royal jelly intake in HDU-treated rats successfully improved sperm quality, hormonal and antioxidant status, and reproductive organ histoarchitecture. Thus, it could be concluded that royal jelly is endowed with antioxidative and anti-inflammatory activities and could be, therefore, used as an adjuvant remedy to improve HDU-induced male subfertility.

Establishment of a rapid drug screening system based on embryonic stem cells.

Embryonic stem (ES) cells have the capacity for self-renewal and differentiation into three germ layers following formation of embryonic bodies (EB). To investigate toxicity of pharmaceutical compounds, five toxic chemicals, indomethacin, dexamethasone, hydroxyurea, 5-fluorouracil, and cytosine arabinoside were applied in mouse ES cells during formation of EBs. Using microscopic evaluation, the size of EBs was reduced in a dose-dependent manner by treatment with pharmaceutical chemicals. While apoptosis-related proteins, cleaved caspase-3 and PARP, were decreased in compound-exposed EBs, necrosis-related protein (Hmgb1) was present in culture media of EBs, indicating that detection of Hmgb1 can result in activation of necrosis by pharmaceutical compounds. While pharmaceutical compounds impaired the differentiation of mES cells linked with spontaneous apoptotic cell death, it was determined that cytotoxic cell damage is necrosis-dependent in mES cells. In addition, an apoptotic transcript (Noxa mRNA) in toxicant-exposed EBs was decreased in parallel with apoptosis-related proteins. Following impairment of apoptosis, differentiation-related markers including un-differentiation (Sox2), endoderm (Hnf4), mesoderm (Bmp4), and ectoderm (Pax6) also fluctuated by treatment with pharmaceutical compounds. Taken together, the data imply that exposure to pharmaceutical compounds results in increased cell death hindering the spontaneous apoptosis of cells to undergo differentiation. Using both characteristics of ES cells like self-renewal or cellular pluripotency and potentials of ES cells for evaluation in toxicity of various compounds, the current study was conducted for establishment of a novel drug screening system beyond hidden virtues of the well-known chemicals.

Spirulina maxima and its protein extract protect against hydroxyurea-teratogenic insult in mice.

Congenital malformations are one of the major causes of child mortality all over the world. In order to prevent them it is necessary to find substances that act as anti-teratogenic agents. In this study hydroxyurea (HU), an antineoplastic and teratogenic drug, was administered to pregnant mice because one of its major mechanisms of teratogenesis is the production of reactive oxygen species (ROS). The aim of this work was to determine if Spirulina maxima (SP) and its aqueous protein extract could protect against HU-teratogenic insult in mouse embryos. SP has been used for a long time because of its nutritional and pharmacological properties. The antioxidant activity, one of the most important, is related to the protein extract due to its content of phycobiliproteins. It was observed that neither SP nor its extract provoked teratogenic effects at any dose tested and even increased vitelline yolk sac circulation. Dams exposed to HU (30 mg/kg, i.p.) presented embryos with multiple alterations in their development. Groups treated with SP or its extract, before and after HU exposure, showed a protector effect in a dose-dependent manner. TBARS test confirmed that the protection effect was related to the antioxidant activity of both SP and its extract.

The impact of human superoxide dismutase 1 expression in a mouse model on the embryotoxicity of hydroxyurea.

BACKGROUND: Oxidative stress is hypothesized to mediate embryotoxicity during organogenesis, yet the reactive oxygen species involved are not defined. The superoxide oxygen radical is converted to hydrogen peroxide, a less reactive species, by superoxide dismutases (SODs). If superoxide is important in mediating embryotoxicity, increased SOD expression should protect embryos against insult. Exposure to hydroxyurea during organogenesis causes brain defects, cleft palate, tail anomalies, and limb defects; administration of D-mannitol, a free radical scavenger, ameliorates hydroxyurea embryotoxicity, suggesting that oxidative stress is important. To elucidate the role of superoxide in mediating hydroxyurea embryotoxicity, we assessed the impact of human SOD1 expression in a murine model. METHODS: hSOD1 hemizygous male mice, carrying the human SOD1 gene, were mated to wild-type or hSOD1 hemizygous females. Dams were treated on gestation day (GD) 9 with saline (control) or 400 (low) or 600 (high) mg/kg hydroxyurea (n = 8-13/group). Mice were euthanized on GD 18 and developmental toxicity was assessed. RESULTS: Exposure to hydroxyurea caused a dose-dependent increase in fetal deaths that was not affected by hSOD1 expression; hydroxyurea decreased fetal weights in litters from wild-type but not hemizygous dams. Hydroxyurea increased the incidence of external and skeletal malformations; fetuses from hemizygous dams treated with high-dose hydroxyurea had fewer malformations compared to wild-type dams. There was no correlation between embryonic phenotype and genotype or SOD activity. CONCLUSION: Maternal hSOD1 expression protected fetuses against malformations induced by hydroxyurea, providing evidence that superoxide plays a role in mediating the response of organogenesis stage embryos to this teratogen.

Sensitivity of RECQL4-deficient fibroblasts from Rothmund-Thomson syndrome patients to genotoxic agents.

RECQ helicase protein-like 4 (RECQL4) is a member of the human RECQ family of DNA helicases. Two-thirds of patients with Rothmund-Thomson syndrome (RTS) carry biallelic inactivating mutations in the RECQL4 gene. RTS is an autosomal recessive disorder characterized by poikiloderma, sparse hair, small stature, skeletal abnormalities, cataracts, and an increased risk of cancer. Mutations in two other RECQ helicases, BLM and WRN, are responsible for the cancer predisposition conditions Bloom and Werner syndromes, respectively. Previous studies have shown that BLM and WRN-deficient cells demonstrate increased sensitivity to hydroxyurea (HU), camptothecin (CPT), and 4-nitroquinoline 1-oxide (4NQO). Little is known about the sensitivity of RECQL4-deficient cells to these and other genotoxic agents. The purpose of this study was to determine if RTS cells display any distinct cellular phenotypes in response to DNA damaging agents or replication blocks that could provide insight into the molecular function of the RECQL4 protein. Our results show that primary fibroblasts from RTS patients carrying two deleterious RECQL4 mutations, compared to wild type (WT) fibroblasts, have increased sensitivity to HU, CPT, and doxorubicin (DOX), modest sensitivity to other DNA damaging agents including ultraviolet (UV) irradiation, ionizing radiation (IR), and cisplatin (CDDP), and relative resistance to 4NQO. The RECQ family of DNA helicases has been implicated in the regulation of DNA replication, recombination, and repair. Because HU, CPT, and DOX exert their effects primarily during S phase, these results support a greater role for the RECQL4 protein in DNA replication as opposed to repair of exogenous damage.

Fisher information matrix of the Dirichlet-multinomial distribution.

In this paper we derive explicit expressions for the elements of the exact Fisher information matrix of the Dirichlet-multinomial distribution. We show that exact calculation is based on the beta-binomial probability function rather than that of the Dirichlet-multinomial and this makes the exact calculation quite easy. The exact results are expected to be useful for the calculation of standard errors of the maximum likelihood estimates of the beta-binomial parameters and those of the Dirichlet-multinomial parameters for data that arise in practice in toxicology and other similar fields. Standard errors of the maximum likelihood estimates of the beta-binomial parameters and those of the Dirichlet-multinomial parameters, based on the exact and the asymptotic Fisher information matrix based on the Dirichlet distribution, are obtained for a set of data from Haseman and Soares (1976), a dataset from Mosimann (1962) and a more recent dataset from Chen, Kodell, Howe and Gaylor (1991). There is substantial difference between the standard errors of the estimates based on the exact Fisher information matrix and those based on the asymptotic Fisher information matrix.

Hydroxyurea-induced toxic side-effects in animals and an attempt at reducing them with vitamins E and C.

Using the changes in several blood parameters and in the histological picture of the liver as markers of toxicity, the effect of hydroxyurea (HU) in healthy rabbits or mice was examined during two weeks after a single administration of this drug. In rabbits a transient significant decrease in blood erythrocyte count with a gradual increase in their osmotic resistance, a suppression of granulocyte phagocytic capacity, and an elevation of acid phosphatase activity in the serum were found. An increase in the proportion of lymphocytes without detectable lysosomes as tested by supravital staining also appeared. No significant difference was observed in the white cell count and lipoperoxide levels after HU administration. Histological picture of the liver, excised from HU-treated mice, indicated a marked hepatotoxicity of the drug. Some of the toxic effects were reduced in animals supplemented with vitamins E and C.

Analysis of the activity of DNA, RNA, and protein synthesis inhibitors on Xenopus embryo development.

The teratogenic and growth-inhibiting potential of DNA, RNA, and protein synthesis inhibitors was explored using the Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Endpoints measured in 96-h static tests were survival, malformation, ability to swim, skin pigmentation, stage of development, and growth. The DNA synthesis inhibitors hydroxyurea, cytosine arabinoside, and ethidium bromide proved to be teratogenic by the severity of malformations induced. Hydroxyurea gave an LC50 of 1.82 mg/ml, an EC50 (malformation) of 0.43 mg/ml, while the values for cytosine arabinsode were 5.41 and 0.76, respectively. The values for ethidium bromide were 0.05 and 0.035. The RNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide were more embryolethal than teratogenic but significantly inhibited growth as determined by head-tail length measurements. Actinomycin D caused severe malformations, while cycloheximide caused relatively minor abnormalities. The LC50 for actinomycin D was 1.89 mg/ml, while the EC50 (malformation) was 2.17 mg/ml. For cycloheximide, the values were 1.59 and 1.19, respectively. FETAX advantages include rapid data collection, the ability to measure stage-dependent effects, and the ability to use a large number of embryos to obtain excellent dose-response curves with narrow confidence limits. Disadvantages include lack of a metabolic activation system, absence of a placental relationship, and the inability to detect specific abnormalities such as limb defects in 96 h.

A teratogenicity study on hydroxyurea and diphenylhydantoin in cats.

Hydroxyurea, an antitumor drug and known teratogen in rat, miniature swine and dog, and diphenylhydantoin, a teratogen in mouse and rat, were assessed for teratogenic effects in cat. Pregnancies were induced, by synchronizing gonadotropin-stimulated estrus and ovulation with natural copulations. Hydroxyurea at 50 or 100 mg/kg, and sodium diphenylhydantoin at 1 or 2 mg/kg dosages, were administered orally in single daily doses from gestation days 10-22. Appropriate controls given empty capsules, were included for each drug. Cats were necropsied on gestation day 43. Fetuses were examined for external, visceral and skeletal malformations. Hydroxyurea at 50 mg/kg dose produced a low teratogenic activity and at 100 mg/kg a high incidence of non-pregnancy and resorptions with, consequently, fewer live fetuses. Diphenylhydantoin gave no clear evidence of teratogenicity at any test dose but was embryolethal at the maternally toxic dose of 2 mg/kg. So far, studies conducted suggest that the cat is a useful species for screening drugs and chemicals for their teratogenic potential.