Difference in metabolic profile of potassium canrenoate and spironolactone in the rat: Mutagenic metabolites unique to potassium canrenoate

The metabolic fates of potassium canrenoate (PC) and spironolactone (SP) were compared for the rat in vivo and in vitro. Approximately 18% of an in vivo dose of SP was metabolized to canrenone (CAN) and related compounds in the rat. In vitro, 20–30% of SP was dethioacetylated to CAN and its metabolites by rat liver 9000 g supernatant (S9). Thus, the major route of SP metabolism is via pathways that retain the sulfur moiety in the molecule. PC was metabolized by rat hepatic S9 to 6, 7- and 6, 7-epoxy-CAN. The -epoxide was further metabolized to its 3- and 3-hydroxy derivatives as well as its glutathione (GSH) conjugate. Both 3- and 3-hydroxy-6, 7-epoxy-CAN were shown to be direct acting mutagens in the mouse lymphoma assay, whereas 6, 7- and 6, 7-epoxy-CAN were not. These mutagenic metabolites, their precursor epoxides and their GSH conjugates were not formed from SP under identical conditions. The above findings appear to be due to inhibition of metabolism of CAN formed from SP by SP and/or its S-containing metabolites, since the in vitro metabolism of PC by rat hepatic microsomes was appreciably reduced in the presence of SP. The hypothesized mechanism(s) for this inhibition is that SP and its S-containing metabolites specifically inhibit an isozyme of hepatic cytochrome P-450 or SP is a preferred substrate over PC/CAN for the metabolizing enzymes. Absence of the CAN epoxide pathway in the metabolism of SP provides a possible explanation for the observed differences in the toxicological profiles of the two compounds.     

DNA damage in leukocytes of mice treated with copper sulfate

Single stranded DNA breaks induced by copper sulfate (CuSO₄) in mice has been studied in vivo using Alkaline Single Cell Gel Electrophoresis (Comet assay). Mice were administered orally with doses of 0, 1.25, 2.50, 5.00, 7.50, 10.00 and 12.50 mg/kg body weight (b.wt.) of CuSO₄ respectively. The samples of whole blood were collected at 24, 48, 72 h, first week and second week post-treatment and the assay was carried out to determine single strand DNA breaks as represented by comet tail-length. In addition, the sample was used to study the repair efficiency by incubating the samples with RPMI medium for 2 h. Results indicated a significant DNA damage at all the doses after treatment with CuSO₄ when compared to controls showing a clear dose-dependent response (p < 0.05). A gradual decrease in the tail-lengths from 48 h post-treatment was observed and by second week, the values returned to control levels at all doses. The study on the repair efficiency indicated that mice treated with all the doses of CuSO₄ showed decrease in mean comet tail-length indicating repair efficiency capacity but less when compared to those of controls. The study also reveals that comet assay is a sensitive and rapid method for detecting DNA damage caused by trace metals such as copper (Cu).     

ATP敏感钾通道亚单位在大鼠组织中的表达

目的　研究ATP敏感钾通道 (KATP)亚单位在大鼠组织中的表达。方法　逆转录多聚酶链式反应 (RT PCR)检测通道mRNA的表达。结果　左心室有Kir 6 1,Kir 6 2和SUR 2A的表达 ,大脑皮层中Kir 6 1,Kir 6 2 ,SUR 1,SUR2B均有表达 ,主动脉平滑肌有Kir 6 1,Kir 6 2 ,SUR 2B的表达 ,膀胱平滑肌中有Kir 6 1,Kir 6 2 ,SUR 2B的表达。结论　各组织的KATP组成有所不同 ,Kir 6 1、Kir 6 2和SUR 2B亚单位在多组织中广泛表达。     

DNA damage in deoxynucleosides and oligonucleotides treated with peroxynitrite.

Peroxynitrite (ONOO-) is a powerful oxidizing agent that forms in a reaction of nitric oxide (NO*) and superoxide (O2-*). We have investigated ONOO--induced DNA damage using deoxynucleosides and oligonucleotides as model substrates, with particular attention paid to the oxidation of 8-oxodG by ONOO-. With regard to deoxynucleosides, ONOO- was found to have significant reactivity only with dG; dA, dC, and dT showed minimal reactivity. However, two of the major products of ONOO--induced oxidation of dG (8-oxodG and 8-nitroG) were both found to be significantly more reactive with ONOO- than with dG. In the context of an oligonucleotide, we observed a concentration-dependent oxidation of 8-oxodG to at least two types of products, one appearing at ONOO- concentrations of /=500 microM. We also examined the susceptibility of these oxidation products to repair by FaPy glycosylase, endonuclease III, uracil glycosylase, and MutY. FaPy glycosylase, which recognizes 8-oxoG as its primary substrate, was the only enzyme that exhibited an efficient reaction with 8-oxodG oxidation products at low ONOO- concentrations (/=500 microM either was not recognized or was poorly repaired by the enzymes. While processing of the lesions was inefficient with endonuclease III and not apparent with uracil glycosylase, the excision of A opposite an 8-oxoG lesion by the enzyme MutY was not affected by the reaction of 8-oxoG with ONOO-. In addition to demonstrating the complexity of ONOO- DNA damage chemistry, these results suggest that 8-oxodG may be a primary target of ONOO- in DNA.     

Failure of potassium canrenoate to alter experimentally induced digitalis arrhythmias

The antiarrhythmic effects of potassium canrenoate were examined under in vivo and in vitro conditions. In the pentobarbital anesthetized dog, ventricular tachycardia was produced by the i.v. administration of ouabain, 64.4 ± 3.7 μg/kg. Potassium canrenoate was administered in increments of 10 mg/kg until a total dose of 30 mg/kg had been achieved. 1 animal developed ventricular fibrillation and in the remaining 7 dogs, the ouabain-induced ectopic focus was not suppressed. However, in 3 dogs, a sinus or junctional tachycardia occured after potassium canrenoate was administered and the rates exceeded that of the ventricular focus. Stimulation of the distal right vagus, however, resulted in a suppression of the supraventricular rhythm and a reappearance of the ventricular ectopic focus. In the rabbit perfused isolated heart, potassium canrenoate (10^?3 M) failed to prevent or reverse acetylstrophanthidin-induced arrhythmias. It is concluded that potassium canrenoate fails to suppress or reverse the cardiotoxic effects of digitalis in the intact dog heart or in isolated heart muscle preparations. In addition, sodium canrenoate does not prevent the positive inotropic action of ouabain.     

Antigenotoxic effect of the Plumbago zeylanica extract against the genotoxic damage induced by potassium canrenoate in cultured human peripheral blood lymphocytes

In India, natural preparations derived from the plants are widely use for the treatments of various diseases. Hence, it becomes necessary to assess the modulating action of the plant extract when associated with other substances. Potassium canrenoate (PC) is a synthetic steroid and is used in the treatment of hypertension. It is not only a genotoxic agent, but also a tumor-initiating agent. In the present study, the effect of various doses (i.e., 5, 10, 20, and 30 µM) of PC were studied for their genotoxic effects in the presence of S9 mix in cultured human lymphocytes, using mitotic index, chromosomal aberrations, sister chromatid exchanges, and replication index as parameters. PC was found to be genotoxic at 20 and 30 µM. Treatment of 30 µM of PC was given along with different doses of Plumbago zeylanica extract (i.e., 107.5, 212.5, 315, and 417 µg/mL) of the culture medium. A dose-dependent decrease in the genotoxic effects of PC was observed. The result suggested that the plant extract per se does not have genotoxic potential, but can modulate the genotoxicity of PC in cultured human lymphocytes.     

Antigenotoxic effect of the Plumbago zeylanica extract against the genotoxic damage induced by potassium canrenoate in cultured human peripheral blood lymphocytes

In India, natural preparations derived from the plants are widely use for the treatments of various diseases. Hence, it becomes necessary to assess the modulating action of the plant extract when associated with other substances. Potassium canrenoate (PC) is a synthetic steroid and is used in the treatment of hypertension. It is not only a genotoxic agent, but also a tumor-initiating agent. In the present study, the effect of various doses (i.e., 5, 10, 20, and 30 μM) of PC were studied for their genotoxic effects in the presence of S9 mix in cultured human lymphocytes, using mitotic index, chromosomal aberrations, sister chromatid exchanges, and replication index as parameters. PC was found to be genotoxic at 20 and 30 μM. Treatment of 30 μM of PC was given along with different doses of Plumbago zeylanica extract (i.e., 107.5, 212.5, 315, and 417 μg/mL) of the culture medium. A dose-dependent decrease in the genotoxic effects of PC was observed. The result suggested that the plant extract per se does not have genotoxic potential, but can modulate the genotoxicity of PC in cultured human lymphocytes.     

Comparative clinical study of spironolactone and potassium canrenoate. A randomized evaluation with double cross-over

In the present study 54 cirrhotic patients were investigated in order to compare the clinical effects of spironolactone (100-200 mg/d) and potassium canrenoate (50-200 mg/d). Diagnosis was established on clinical and laboratory findings for at least 12 months and/or on liver biopsy: no patients had signs of hepatic decompensation. Patients entering the study without previous treatment, after a basal period of observation, were randomly allocated to one of the spirolactones (spironolactone or potassium canrenoate); those already under antialdosteronic treatment underwent a first observation period and were then all shifted to the other drug. After completing a second observation all patients underwent a second cross-over and a new assessment of clinical and laboratory parameters after the third period of observation. 31 patients completed all observations (3.5 months each, overall mean). No differences in liver function tests were present during follow-up. Maintenance of body weight was achieved with a dose of potassium canrenoate half that of spironolactone. Serum K+ was increased in each patient after spironolactone and potassium canrenoate as compared to the basal period. The 24-h urinary excretion of K+ was significantly decreased in each patient after both drugs. No significant changes were observed in both Na+ and Cl- plasmatic concentration and urinary excretion. Gynaecomastia was present in 3/11 patients during the basal observation, in 13/30 patients under spironolactone and in 5/25 patients under potassium canrenoate; this finding, however, was not correlated to changes in the basal serum concentration of prolactin.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA damage in normal and neoplastic mouse tissues after treatment with misonidazole in vivo

Alkaline elution has been used to examine the integrity of DNA isolated from various tissues from mice treated with misonidazole (MISO). High doses (1-3 mg/g) of MISO caused extensive DNA strand breakage in cells isolated from two fibrosarcoma tumors that were known to contain hypoxic cells, and also in cells from certain normal tissues (liver and kidney in particular). The incidence of strand breaks gives further support to the suggestion that MISO can be metabolically nitroreduced beyond the singly reduced nitro radical-anion in some normal tissues as well as in hypoxic tumor cells, generating DNA-reactive species. Nitroreductases must therefore be able to compete successfully with molecular oxygen for the MISO nitro radical-anion in such tissues.     

Oxidative DNA damage in XPC-knockout and its wild mice treated with equine estrogen

Long-term hormone replacement therapy with equine estrogens is associated with a higher risk of breast, ovarian, and endometrial cancers. Reactive oxygen species generated through redox cycling of equine estrogen metabolites may damage cellular DNA. Such oxidative stress may be linked to the development of cancers in reproductive organs. Xeroderma pigmentosa complementation group C-knockout ( Xpc-KO) and wild-type mice were treated with equilenin (EN), and the formation of 7,8-dihydro-8-oxodeoxyguanosine (8-oxodG) was determined as a marker of typical oxidative DNA damage, using liquid chromatography electrospray tandem mass spectrometry. The level of hepatic 8-oxodG in wild-type mice treated with EN (5 or 50 mg/kg/day) was significantly increased by approximately 220% after 1 week, as compared with mice treated with vehicle. In the uterus also, the level of 8-oxodG was significantly increased by more than 150% after 2 weeks. Similar results were observed with Xpc-KO mice, indicating that Xpc does not significantly contribute to the repair of oxidative damage. Oxidative DNA damage generated by equine estrogens may be involved in equine estrogen carcinogenesis.     

DNA damage in organs of mice treated acutely with patulin, a known mycotoxin

Patulin, a known mycotoxin, is considered a significant contaminant in apples, apple-derived products and feeds. This study investigated the genotoxic effects of patulin in multiple organs (brain, kidney, liver and urinary bladder) of mice using an in vivo comet assay. We assessed the mechanism underlying this genotoxicity by measuring the GSH content and the thiobarbituric acid-reactive species (TBARS) level. Male CF-1 mice were given 1.0-3.75mg/kg patulin intraperitoneally. The effect of patulin was dose-dependent and the highest patulin dose induced DNA strand breaks in the brain (damage index, DI, in hippocampus increased from 36.2 in control animals to 127.5), liver (44.3-138.4) and kidneys (31.5-99); decreased levels of GSH (hippocampus - from 46.9 to 18.4nmol/mg protein); and an increase in lipid peroxidation (hippocampus - from 5.8 to 20.3 MDA equivalents/mg protein). This finding establishes an interrelationship between the pro-oxidant and genotoxic effects of patulin. Pre-treatment administration of N-acetyl-cysteine reduced patulin-induced DNA damage (hippocampus - DI from 127.5 to 39.8) and lipid peroxidation (hippocampus - 20.3 to 12.8 MDA equivalents/mg protein) by restoring cellular GSH levels, reinforcing the positive relationship between patulin-induced GSH depletion and DNA damage caused by systemic administration of this mycotoxin.     

Acute effect of potassium canrenoate administration on renin-angiotensin, kallikrein-kinin and prostaglandin systems

1. To investigate the possible effects of potassium canrenoate (PC) on plasma renin activity (PRA) and on renal prostaglandins (PGS) and kinins under elevated sodium and/or potassium intakes, a single dose of PC was administered to four groups of Wistar male rats. 2. They were fed a normal diet (C), a diet supplemented with 4% of NaCl, (Na), with 1% of KCl: (K) or both supplements (NaK). 3. PRA and urinary PGS excretion did not show changes after PC administration, but total urinary kinins showed higher values after the treatment in all groups. 4. A diuretic but not natriuretic effect was observed only in C animals. 5. In conclusion, the single dose of PC was able to stimulate urinary kinins and to spare potassium independently of dietary electrolyte supplements that were able to block the diuretic effect of the drug.     

Population pharmacokinetic model of canrenone after intravenous administration of potassium canrenoate to paediatric patients

AIMS

To characterize the population pharmacokinetics of canrenone following administration of potassium canrenoate to paediatric patients.

METHODS

Data were collected prospectively from 23 paediatric patients (2 days to 10 years of age; median weight 4 kg, range 2.16–28.0 kg) who received intravenous potassium canrenoate (K-canrenoate) as part of their intensive care therapy for removal of retained fluids, e.g. in pulmonary oedema due to chronic lung disease and for the management of congestive heart failure. Plasma samples were analyzed by HPLC for determination of canrenone (the major metabolite and pharmacologically active moiety) and the data subjected to pharmacokinetic analysis using NONMEM.

RESULTS

A one compartment model best described the data. The only significant covariate was weight (WT). The final population models for canrenone clearance (CL/F) and volume of distribution (V/F) were CL/F (l h⁻¹) = 11.4 × (WT/70.0)^0.75 and V/F (l) = 374.2 × (WT/70) where WT is in kg. The values of CL/F and V/F in a 4 kg child would be 1.33 l h⁻¹ and 21.4 l, respectively, resulting in an elimination half-life of 11.2 h.

CONCLUSIONS

The range of estimated CL/F in the study population was 0.67–7.38 l h⁻¹. The data suggest that adjustment of K-canrenoate dosage according to body weight is appropriate in paediatric patients.     

Dietary catechol causes increased oxidative DNA damage in the livers of mice treated with acetaminophen

We have shown that direct reaction of catechol with nitric oxide (NO) results in generation of reactive oxygen and nitrogen species (RNS) through semiquinone radical formation, leading to oxidative DNA damage in rat forestomach. In the present study, we investigated whether dietary catechol systemically exerts the same effects under NO-rich circumstances, when given before and during induction of inflammatory lesions. Male ICR mice were treated with or without 0.8% catechol in the diet for 2 weeks followed by acetaminophen (APAP) administration at a dose of 300 mg/kg by single i.p. injection. Along with several indicators of APAP-induced hepatitis, 8-hydroxydeoxyguanosine (8-OHdG) levels and immunohistochemistry for 3-nitrotyrosine (NO₂Tyr) in the livers were examined at 1.5, 4 and 24 h after APAP injection. 8-OHdG was significantly increased at 24 h in the co-treatment group, but not with either catechol or APAP alone. Elevation of serum ALT and AST activities, decrease of reduced glutathione levels and histopathological liver changes were observed to the same extents in both APAP-treated groups. In view of the finding of positive hepatocytes for NO₂Tyr prior to generation of 8-OHdG, the process of oxidative DNA damage might involve RNS formation. Precise quantitative analysis of NO₂Tyr by means of liquid chromatography with tandem mass spectrometry (LC-MS/MS) in an additional study with the same experimental protocol confirmed increase of RNS due to the reaction of catechol with NO produced after APAP-induced hepatitis. The overall data imply that antioxidants with a catechol structure can cause oxidative DNA damage under inflammatory conditions.     

Repair kinetics of DNA, RNA and proteins in the tissues of mice treated with doxorubicin

Experiments in mice treated with a single 10 mg/kg dose of doxorubicin (adriamycin) i.p. revealed considerable reduction in the incorporation of 3H-thymidine in DNA and of 3H-uridine in RNA, in the spleen and liver, and at mitochondrial and non-mitochondrial level in the heart. Although protein syntheses in the heart and spleen were not reduced by the drug to any great degree, they took 10 days to return to normal; conversely, liver protein syntheses were not inhibited at all and indeed presented signs of stimulation.