Todralazine protects zebrafish from lethal effects of ionizing radiation: role of hematopoietic cell expansion.

The Johns Hopkins Clinical Compound Library (JHCCL), a collection of Food and Drug Administration (FDA)-approved small molecules (1400), was screened in silico for identification of novel ß2AR blockers and tested for hematopoietic stem cell (HSC) expansion and radioprotection in zebrafish embryos. Docking studies, followed by the capacity to hasten erythropoiesis, identified todralazine (Binding energy, -8.4 kcal/mol) as a potential HSC-modulating agent. Todralazine (5 µM) significantly increased erythropoiesis in caudal hematopoietic tissue (CHT) in wild-type and anemic zebrafish embryos (2.33- and 1.44-folds, respectively) when compared with untreated and anemic control groups. Todralazine (5 µM) treatment also led to an increased number of erythroid progenitors, as revealed from the increased expression of erythroid progenitor-specific genes in the CHT region. Consistent with these effects, zebrafish embryos, Tg(cmyb:gfp), treated with 5 µM todralazine from 24 to 36 hours post fertilization (hpf) showed increased (approximately two-folds) number of HSCs at the aorta-gonad-mesonephros region (AGM). Similarly, expression of HSC marker genes, runx1 (3.3-folds), and cMyb (1.41-folds) also increased in case of todralazine-treated embryos, further supporting its HSC expansion potential. Metoprolol, a known beta blocker, also induced HSC expansion (1.36- and 1.48-fold increase in runx1 and cMyb, respectively). Todralazine (5 µM) when added 30 min before 20 Gy gamma radiation, protected zebrafish from radiation-induced organ toxicity, apoptosis, and improved survival (80% survival advantage over 6 days). The 2-deoxyribose degradation test further suggested hydroxyl (OH) radical scavenging potential of todralazine, and the same is recapitulated in vivo. These results suggest that todralazine is a potential HSC expanding agent, which might be acting along with important functions, such as antioxidant and free radical scavenging, in manifesting radioprotection.

A novel mechanism for drug-induced liver failure: inhibition of histone acetylation by hydralazine derivatives.

BACKGROUND/AIMS: The aim of this study was to investigate the precise mechanism of liver failure by hydralazine derivatives, with special reference to liver regeneration failure. METHODS: Histone acetylation and proliferation of hepatocytes were evaluated by immunohistochemistry with anti-acetylated histone H4 and proliferating cell nuclear antigen (PCNA). Inhibition of histone acetylation by drugs was determined by in vitro histone acetylation assay. Mice livers fed with todralazine for 1 or 4 months were subjected to immunohistochemistry and Western blotting. Todralazine-fed mice were challenged with anti-Fas to check liver regeneration failure. RESULTS: On immunohistochemistry, histone acetylation in the hepatocytes was significantly impaired in patients with hydralazine derivatives. In an in vitro acetyl transferase assay, histone acetylation was inhibited by hydralazine derivatives in a dose-dependent manner. Mice fed with todralazine (3mg/day) for 4 months showed impairment of histone acetylation in hepatocytes whereas no inhibition was observed in mice fed with todralazine for 1 month. Anti-Fas challenge to todralazine-fed mice resulted in impairment of liver regeneration in respect of liver weight loss with impairment of histone acetylation in hepatocytes. CONCLUSIONS: Todralazine could inhibit catalysis of histone acetyltransferase and long-term administration of todralazine may impair histone acetylation of the hepatocytes, resulting in liver regeneration failure.

DNA repair of hydrogen peroxide-induced damage in human lymphocytes in the presence of four antimutagens. A study with alkaline single cell gel electrophoresis (comet assay).

We assessed four antimutagenic compounds' influences on DNA repair in human lymphocytes exposed in vitro to hydrogen peroxide (20 microM, 5 min, at 4 degrees C). DNA damage and repair were estimated by means of alkaline single cell gel electrophoresis (comet assay). It was noticed that the enhancement of DNA repair was relatively strongest when fluphenazine was present in the cell culture medium. In the cases of anthocyanins and alkylresorcinols, the effects were almost 6-9 times weaker than that of FPh. The effect of todralazine on DNA repair was relatively weakest. Further study should be done on fluphenazine as a potential DNA repair-enhancing compound.

Evaluation of antimutagenic effect of todralazine in cultured lymphocytes.

Todralazine, an antihypertensive drug from the hydrazinophthalazine group, significantly decreased the activities of benzo[a]pyrene and mitomycin C in three short-term genotoxicity tests in human lymphocyte cultures. The thioguanine resistance test, the cytokinesis-blocked micronucleus assay and the sister chromatid exchange test were used to demonstrate the antimutagenicity of todralazine. Todralazine lowered the level of free radicals generated by human granulocytes in vitro in the presence of benzo[a] pyrene and also in the presence of the granulocyte activator and tumor promoter phorbol myristate acetate. These results, together with our previous data obtained in the standard bacterial Ames test, strongly suggest that todralazine is a good antimutagen in vitro and deserves further research on its inhibitory action on mutagenesis and carcinogenesis.

Inhibition of potassium dichromate mutagenicity by todralazine.

Todralazine, an antihypertensive drug of the hydrazinoph-thalazine group, markedly decreased the mutagenic activity of potassium dichromate in standard bacterial tests. At the highest todralazine dose tested inhibition of potassium dichromate mutagenic activity by approximately 90% in the Ames test and up to 100% (complete) inhibition in the Bacillus subtilis rec- assay was observed. Spectrophotometric analyses proved that todralazine induced reduction of Cr(VI) to Cr(III) and complexation of Cr(III) ions. These spectro-photometric results may be a presumptive explanation of the observed mutagenic activity decrease, as it is known that Cr(III) is poorly transported across cell membranes and therefore is not mutagenic to bacterial cells. We perceive our experiments as an example of attempts which should lead to an effective reduction in chromium genotoxic and carcinogenic activity in exposed individuals.

Genotoxicity of hydrazino-phtalazine antihypertensive drugs assessed by an in vitro micronucleus assay.

The genotoxicity of antihypertensive drugs, hydralazine, dihydralazine and binazine was assessed on the base of their capacity to induce micronuclei in L929 cell line. In our previous investigations we indicated that these drugs did not induce micronuclei in bone marrow polichromatic erythrocytes in PZH SFISS mice. Present results show that hydralazine and dihydralizine can induce micronuclei in vitro and that this effect depends on time of exposure and the concentration of drug.

Benidipine improves endothelial function in renal resistance arteries of hypertensive rats.

We studied the effects of long-term antihypertensive treatment on endothelial function in renal resistance arteries from spontaneously hypertensive rats (SHR). Wistar-Kyoto rats (WKY) were used as a normotensive reference. Adult SHR were treated with benidipine (a calcium antagonist) or ecarazine (a vasodilator) for 10 weeks; the drugs caused similar reductions in blood pressure. Changes in isometric tension of rings prepared from the third-order branches of the renal arteries were recorded. Endothelium-dependent relaxations induced by acetylcholine in rings contracted with norepinephrine were smaller in SHR than in WKY. The impaired relaxation was improved by benidipine treatment, but ecarazine had no significant effect. In vitro treatment with meclofenamic acid, a cyclooxygenase inhibitor, did not alter the differences in the relaxations. In the presence of meclofenamic acid, N omega-nitro-L-arginine methyl ester slightly reduced the relaxations; the relaxation was smaller in SHR than in WKY and was not affected by benidipine treatment. In rings contracted with 40 mmol/L. KCI, the relaxations induced by acetylcholine in the presence of meclofenamic acid were smaller than those in rings contracted with norepinephrine. The relaxation was smaller in SHR than in WKY but was normalized by benidipine treatment. Thus, acetylcholine relaxes rat renal resistance arteries by releasing nitric oxide and endothelium-derived hyperpolarizing factor from the endothelium, which is impaired in SHR. Long-term benidipine treatment improves the impaired relaxation in SHR by enhancing nitric oxide-mediated relaxation.

Fulminant hepatic failure caused by ecarazine hydrochloride (a hydralazine derivative).

The cause of fulminant hepatic failure is reported to be unknown in more than half the cases in Japan. We recently reviewed 23 cases of fulminant hepatic failure that had been treated at our hospital. The cause of disease had been regarded as unknown before this study. It was found that seven of these patients had been under ecarazine hydrochloride therapy when they developed fulminant hepatic failure. We examined the reasons why fulminant hepatic failure in these seven patients had not been previously attributed to ecarazine, and found that it could be explained by the following factors: (1) the time from the start of ecarazine therapy to the onset of hepatic failure was long; (2) in all cases, hepatic failure developed more than 10 days after the clinical recognition of hepatitis; and (3) characteristic signs of drug-induced hepatic failure such as a skin rash and positive lymphocytes stimulation test with the drug were absent in all cases. Fulminant hepatic failure in these cases could be characterized by: (1) rapid decrease in serum alanine transaminase (ALT) level after discontinuation of ecarazine, (2) prolonged jaundice despite discontinuation of ecarazine, (3) high incidence of anti-nuclear antibody (ANA) (57%), and (4) histological findings of extensive hepatocellular necrosis ranging from bridging necrosis to massive necrosis. Of the seven patients, four died of fulminant hepatic failure. These four patients had received high doses of ecarazine hydrochloride for prolonged periods. Our data suggest that there may be many cases in which the cause of fulminant hepatic failure or acute hepatitis was not previously determined that can be attributed to long-term drug therapy for chronic diseases.

Application of arylosulfonic acids in analysis of antihypertensive drugs.

Suitability of four arylosulfonic acids and their sodium salts to form derivatives with hypotensive drugs were studied. New crystalline arylosulfonates of todralazine, hydralazine and dihydralazine were obtained. Physico-chemical properties of the obtained arylosulfonates were tested. Reagents mentioned above were also used in analysis of these drugs.

Genetic effects of binazine and hydralazine in vitro and in vivo.

The mutagenic and genotoxic activities of binazine and hydralazine were studied. In the Ames test, both with and without S-9 fraction, hydralazine was mutagenic in strains Salmonella typhimurium TA100 and TA1537, whereas binazine was not mutagenic in these strains. Both drugs were negative in mice micronucleus test.

Evaluation of the mechanisms of todralazine effect on mutagenicity of benzo(a)pyrene.

We have previously described that todralazine markedly decreased mutagenicity of several indirect- and direct-acting mutagens. In this paper we report the results of experiments conducted in order to evaluate the involvement of desmutagenic and bio-antimutagenic activities in the observed antimutagenic effect of todralazine. The results of the Ames test suggest a bio-antimutagenic, and not desmutagenic effect of todralazine. The separation of B(a)P and their derivatives by thin layer chromatography, performed after in vitro incubation of this promutagen with S9 fraction and todralazine revealed almost complete decline of B(a)P derived products in the presence of todralazine. The results indicate that the observed antimutagenic effect of todralazine on B(a)P mutagenicity is bio-antimutagenic rather than desmutagenic in their nature.

Todralazine influence upon the mutagenicity of some direct- and indirect-acting mutagens.

Todralazine decreased the mutagenic activity of tested direct- and indirect-acting mutagens. Despite the marked differences between efficient todralazine doses (ED50) it was observed that, in the case of tested indirect mutagens as well as in some of the direct mutagens, the decrease of mutagenicity by todralazine was very strong, exceeding 80% in some cases.

Joint determination of todralazine and acetazolamide in human serum by differential pulse polarography.

Differential pulse polarography (DPP) is proposed as a direct method for the quantitation of todralazine and acetazolamide in human serum. The method was applied to the determination of these drugs in human serum, after a liquid-liquid extraction process. This extraction process together with the use of the standard additions method is essential for the elimination of the matrix effect. The proposed method enables detection limits of 0.107 microgram ml-1 for acetazolamide and 0.111 microgram ml-1 for todralazine to be achieved at reduction potentials of -0.59 and -0.86 V, respectively, using Britton-Robinson buffer (pH 1.65) as the supporting electrolyte.

Study of the mechanism of todralazine antimutagenic activity: the effect upon mutagenicity of daunorubicine.

Todralazine markedly reduced the mutagenic activity of the standard direct-acting mutagen--daunorobicine (DRC)--in the Ames test. Spectrophotometric measurements proved that todralazine did not interact with DRC in water solution. Todralazine neither interacted with calf thymus DNS in vitro, nor changed the interaction of DRC with DNA. Therefore we concluded that the decrease of DRC mutagenicity observed in the Ames test should be explained rather in terms of a bioantimutagenic than a desmutagenic activity of todralazine.

Mutagenicity of B(a)P in the presence of some hydralazine derivatives.

Hydralazine, dihydralazine and todralazine were tested in the aspect of their mutagenic potency, and the influence upon the mutagenicity of standard promutagen--B(a)P. Hydralazine exhibited strong mutagenic activity in the Ames test while mutagenic activity of dihydralazine was relatively weak. Todralazine had no mutagenic activity, and significantly decreased mutagenicity of B(a)P. It was concluded that todralazine could be a good antimutagenic substance.

Acute systemic toxicity and antihypertensive activity of a novel todralazine analog in rats.

Acute intravenous toxicity and antihypertensive activity of KB1, a novel todralazine analog was investigated and compared with the effects of todralazine (Td) in normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. LD50 values were 72 mg.kg-1 for KB1, and 255 mg.kg-1 for Td in WKY and 43 mg.kg-1 or KB1 in SHR. Therefore, the toxicity of KB1 was higher than that of Td and it increased in SHR. The antihypertensive activity of KB1 (ED20% 9.8 mg.kg-1) in WKY was about 9 times less potent in comparison with Td (ED20% 1.1 mg.kg-1). Blood pressure reducing activity of KB1 augmented apparently in SHR (ED20% 2.5 mg.kg-1) whereas Td had not such an effect (ED20% 1.0). Thus, the influence of Td on blood pressure was similar in normotensive and hypertensive animals. Our results indicate that KB1 is capable of reducing blood pressure preferentially in hypertension.

Application of arylosulfonic acids in analysis of antidepressive drugs.

Suitability of three phenylsulphonyloxybenzenesulfonic acids and their sodium salts to form new derivatives with antidepressive drugs were studied. Then physicochemical properties of the obtained arylosulfonates were tested. Reagents mentioned above were also used in analysis of these drugs.

Influence of hydrazinophthalazines on the activity of collagenase in murine fibroblast culture.

Murine fibroblasts cultured in vitro were treated with hydrazinophthalazine drugs inducing the lupus erythematosus-like syndrome. Collagenase enzyme activity in the growth medium was found to be increased 3-fold by hydralazine and 1.8-fold by binazine, as compared to the control cultures. The enhanced level of collagenolytic activity points to the metabolic changes in the connective tissue in response to hydrazinophtalazines.

Biochemical changes in cultured murine fibroblasts after treatment with hydrazinophthalazines.

Fibroblast cultures exposed to the drugs inducing a collagen-like syndrome (hydralazine and binazine) displayed growth inhibition and decrease in cellular protein content in a dose-dependent manner compared with control cultures. This was accompanied by the inhibitory effect of the drugs on DNA synthesis. The changes in the basic biochemical parameters of fibroblasts testify to the toxicity of hydrazinophthalazines in the connective tissue.