Administration Of Cobalt Chloride Research Articles

Study of cobalt doping control via various routes in thoroughbred horses.

Cobalt is a substance that has been abused for athletic performance enhancement and has thus been prohibited by human and animal sports doping control authorities. However, because cobalt is present in humans and animals as a trace element, a certain level of cobalt is naturally present in their excretions. In the racing industry, cobalt is a controlled substance with a threshold concentration specified by the International Agreement on Breeding, Racing and Wagering (IABRW) for international harmonization. Due to environmental and feed consumption differences among countries, regional cobalt concentration trends should be evaluated before cobalt testing is introduced. In this study, we conducted a preliminary evaluation of the urinary concentration of cobalt among a population of racehorses in Korea using inductively coupled plasma mass spectrometry (ICP-MS) analysis, followed by analysis of the urinary release of cobalt after the administration of cobalt chloride in various situations. The normal distribution for the Korea-based racehorses was used to determine a urine concentration limit (96.5ng/ml, risk factor of 1 in 10,000). After the intravenous (IV) administration of CoCl2 , the initial elimination of cobalt was rapid. A high concentration (over 2,000 ng/ml) and a slow excretion pattern were observed during the final 2 weeks of the 3-week observation period. When CoCl2 was administered orally, maximum concentration (Cmax , 92-992 ng/ml) was observed at 6-8h.

Изменение параметров системной гемодинамики на фоне гиперкальциемии в условиях сочетанного и изолированного введения кобальта и цинка

Введение. В организме кальций находится в 3 формах - связанный с белком, главным образом с альбумином, входит в комплекс с бикарбонатом, лактатом, фосфатом и цитратом, и 50% Са в крови находится в ионизированном виде Са++. Цинк является составной частью металлоэнзимов, таких как карбоангидраза, карбоксипептидаза и ДНК-полимераза. Кобальт относится к тем металлам, с которыми кальций взаимодействует в металлоактивных ферментных системах. Установлено, что кобальт может депонироваться в матриксе лизосом в результате образования комплекса с анионными группами и вступать в конкурентную борьбу с ионами Са2+ и Мg2+ за связывание с активными центрами протонной помпы. Цель исследования - изучение особенностей изменения параметров системной гемодинамики на фоне гиперкальциемии в условиях сочетанного и изолированного введения солей тяжелых металлов кобальта и цинка. Методика. Экспериментальную гиперкальциемию у крыс Вистар воспроизводили ежедневным однократным введением 3000 МЕ препарата «Аквадетрим». Хлориды кобальта (4 мг/кг) и цинка (20 мг/кг) вводили внутрижелудочно с помощью зонда ежедневно на протяжении 1 мес. По завершении эксперимента определяли основные параметры системной гемодинамики: артериальное давление инвазивно, путем введения в бедренную артерию катетера; минутный объем крови регистрировали с помощью метода термодилюции, для чего через левую общую сонную артерию в дугу аорты вводился термистор. Рассчитывали по специальным формулам среднее артериальное давление, сердечный индекс, ударный индекс и удельное периферическое сосудистое сопротивление. Результаты. Исследование показало, что при внутрижелудочном введении в течение месяца хлорида кобальта и цинка развивается артериальная гипертензия гипокинетического типа. Экспериментальная гиперкальциемия оказывает некоторое протекторное влияние на развитие кобальтовой интоксикации, а при сочетанном введении кобальта и цинка на фоне гиперкальциемии эффекты металлов усиливаются. Также установлено, что сочетанное введение солей кобальта и цинка вызывает менее выраженный эффект на параметры системной гемодинамики, чем раздельное применение металлов. Заключение. Избыточное поступление хлоридов кобальта и цинка в организм приводит к развитию артериальной гипертензии. Гиперкальциемия может изменять токсические эффекты тяжелых металлов. Сочетанное введение кобальта и цинка оказывает менее выраженный эффект на параметры системной гемодинамики. Calcium belongs to essential macronutrients. In the human body, calcium exists in three forms: bound to proteins, primarily to albumin; as a complex with bicarbonate, lactate, phosphate, or citrate; and as an ionized form of Ca2+ (which accounts for 50% of circulating Ca). Zinc is one of the most important trace elements of the body and an integral part of metalloenzymes, such as carbonic anhydrase, carboxypeptidase, and DNA polymerase. Cobalt belongs to the metals with which calcium interacts in metal-active enzyme systems. Cobalt is able to deposit in the lysosomal matrix by complexation with anionic groups and to compete with Ca2+ and Mg2+ for binding to active centers of the proton pump. The aim of this work was to study changes in parameters of the systemic hemodynamics during hypercalcemia in combined and isolated administration of heavy metal salts of cobalt and zinc. Methods. Experimental hypercalcemia was induced in Wistar rats by intragastrical administration of a water-soluble vitamin D-3 drug Aquadetrim (3,000 IU, daily for one month). Cobalt chloride (4 mg/kg) and zinc chloride (20 mg/kg) were administered intragastrically with a probe, daily for one month. Upon completion of the experiment, the following major indexes of the systemic hemodynamics were determined: blood pressure was measured invasively, with a catheter inserted into the femoral artery, and minute blood volume was recorded using the thermodilution method, with a thermistor introduced into the aortic arch through the left common carotid artery. Mean blood pressure, cardiac index, stroke index, and specific peripheral vascular resistance were calculated using special formulas. Results showed that the monthly intragastric administration of cobalt chloride and zinc chloride was associated with hypokinetic type hypertension. Experimental hypercalcemia provided some protection against cobalt intoxication but the combined administration of the metals during hypercalcemia enhanced their toxicity. The combined administration of cobalt and zinc chloride produced a less pronounced effect on indexes of the systemic hemodynamics than each metal alone. Conclusion. Excessive intake of cobalt chloride and zinc chloride results in arterial hypertension. Hypercalcemia may change the toxic effects of heavy metals. The combined administration of cobalt with zinc produced a less pronounced effect on parameters of the systemic hemodynamics.

Cobalt accumulation in horses following repeated administration of cobalt chloride.

To monitor cobalt concentrations in urine, red blood cells and plasma after chronic parenteral administration of cobalt chloride evaluate these results against the current International Federation of Horseracing Authorities thresholds for detecting cobalt misuse. Eight mares were randomly assigned to four treatment groups, with two mares in each group: Group 1 - control group, Group 2 - 25 milligrams cobalt intravenously as CoCl2 weekly, Group 3 - 50 milligrams cobalt intravenously as CoCl2 weekly, and Group 4 - 25 milligrams cobalt intravenously mid-week and at the end of the week. Urine and blood samples were collected before each weekly administration so that trough levels were assessed. In the group receiving two doses per week, urine and blood were collected prior to the dose given at the end of each week. Samples were initially collected at time zero then weekly for 10 weeks. Three further collections of urine and blood were made at days 81, 106 and 127. Urine creatinine measurements to assess horse hydration status were performed by the Jaffe reaction method. Cobalt determinations in plasma, blood and urine were by inductively coupled plasma-mass spectrometry. Haematocrit concentrations, used to calculate red cell cobalt levels, were performed using a microhematocrit centrifuge. Statistical analyses were conducted in Genstat (v17, VSNi). Marked cobalt accumulation was evident with increasing cobalt concentrations for all sample matrices in specimens collected immediately prior to cobalt administration. Correlation between the sample matrices improved when urine cobalt concentration was adjusted for creatinine level. Red cell cobalt levels remained elevated for at least 12 weeks after cessation of administration, consistent with the lifespan of the red cell. There was no significant change in haematocrit concentrations for the duration of the study. The current urine cobalt threshold was only effective at detecting acute cobalt exposure while the plasma cobalt threshold was able to consistently identify chronic high-level cobalt exposure and potential cobalt misuse. The threshold values legislated for urine cobalt do not correlate with those set for plasma. The acute nature of urinary cobalt excretion provides a relatively small window through which cobalt administration is detected. Plasma and red cell cobalt concentrations can provide a clearer picture of potential cobalt misuse.

Inhibition of HIF-1α restrains fracture healing via regulation of autophagy in a rat model.

It has been demonstrated that bone fracture is associated with the activation of autophagy, and upregulation of autophagy could promote fracture healing. Previous study by our group demonstrated that activating the HIF-1α pathway via administration of cobalt (II) chloride (CoCl2) could promote fracture healing in vivo. However, the role of hypoxia-inducible factor-1α (HIF-1α) in autophagy remains unknown. In the current study, rats were divided into two groups following tibial fracture and treated with echinomycin or dimethyl sulfoxide (DMSO). Rats were sacrificed at 7, 14, 28 and 42 days after fracture. The evaluation of fracture healing was performed by micro-computed tomography. In addition, the effects of echinomycin on microtubule-associated protein 1 light chain 3 (LC3 II), runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), Unc-51-like autophagy activating kinase 1 (ULK1) and P62 were detected at the mRNA and protein levels by reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemistry. The results demonstrated that the expression of LC3 II was markedly decreased following systemic administration of echinomycin (0.05 mg/kg every other day for 42 days, intraperitoneally). Furthermore, the levels of Runx2, ALP and ULK1 were decreased, while those of P62 were increased, at the mRNA and protein levels in rats treated with echinomycin in vivo. In summary, the current study suggested that HIF-1α may serve an important role in fracture healing via the downregulation of autophagy.

Effect of Intravenous Administration of Cobalt Chloride to Horses on Clinical and Hemodynamic Variables.

BackgroundCobalt chloride (CoCl2) is administered to racehorses to enhance performance. The purpose of this study was to evaluate the clinical, cardiovascular, and endocrine effects of parenterally administered CoCl2.ObjectivesTo describe the effects of weekly intravenous doses of CoCl2 on Standardbred horses.AnimalsFive, healthy Standardbred mares.MethodsProspective, randomized, experimental dose‐escalation pilot. Five Standardbred mares were assigned to receive 1 of 5 doses of CoCl2 (4, 2, 1, 0.5, or 0.25 mg/kg) weekly IV for 5 weeks. Physical examination, blood pressure, cardiac output, and electrocardiography (ECG) were evaluated for 4 hours after administration of the first and fifth doses. Blood and urine samples were collected for evaluation of cobalt concentration, CBC and clinical chemistry, and hormone concentrations.ResultsAll mares displayed pawing, nostril flaring, muscle tremors, and straining after CoCl2 infusion. Mares receiving 4, 2, or 1 mg/kg doses developed tachycardia after dosing (HR 60–126 bpm). Ventricular tachycardia was noted for 10 minutes after administration of the 4 mg/kg dose. Increases in systolic arterial pressure (SAP), diastolic arterial pressure (DAP), and mean arterial pressure (MAP) occurred after administration of all doses (4, 2, 1, 0.5, and 0.25 mg/kg). Profound hypertension was observed after the 4 mg/kg dose (SAP/DAP, MAP [mmHg] = 291–300/163–213, 218–279). Hemodynamics normalized by 1–2 hours after administration. ACTH and cortisol concentrations increased within 30 minutes of administration of all CoCl2 doses, and cardiac troponin I concentration increased after administration of the 4 and 2 mg/kg doses.Conclusions and Clinical ImportanceThe degree of hypertension and arrhythmia observed after IV CoCl2 administration raises animal welfare and human safety concerns.

Pharmacokinetics of inorganic cobalt and a vitamin B12 supplement in the Thoroughbred horse: Differentiating cobalt abuse from supplementation

While cobalt is an essential micronutrient for vitamin B12 synthesis in the horse, at supraphysiological concentrations, it has been shown to enhance performance in human subjects and rats, and there is evidence that its administration in high doses to horses poses a welfare threat. Animal sport regulators currently control cobalt abuse via international race day thresholds, but this work was initiated to explore means of potentially adding to application of those thresholds since cobalt may be present in physiological concentrations. To devise a scientific basis for differentiation between presence of cobalt from bona fide supplementation and cobalt doping through the use of ratios. Six Thoroughbred horses were given 10 mL vitamin B12 /cobalt supplement (Hemo-15® ; Vetoquinol, Buckingham, Buckinghamshire, UK., 1.5 mg B12 , 7 mg cobalt gluconate = 983 μg total Co) as an i.v. bolus then an i.v. infusion (15 min) of 100 mg cobalt chloride (45.39 mg Co) 6 weeks later. Pre-and post-administration plasma and urine samples were analysed for cobalt and vitamin B12 . Urine and plasma samples were analysed for vitamin B12 using an immunoassay and cobalt concentrations were measured via ICP-MS. Baseline concentrations of cobalt in urine and plasma for each horse were subtracted from their cobalt concentrations post-administration for the PK analysis. Compartmental analysis was used for the determination of plasma PK parameters for cobalt using commercially available software. On administration of a vitamin B12 /cobalt supplement, the ratio of cobalt to vitamin B12 in plasma rapidly increased to approximately 3 and then rapidly declined below a ratio of 1 and then back to near baseline over the next week. On administration of 100 mg cobalt chloride, the ratio initially exceeded 10 in plasma and then declined with the lower 95% confidence interval remaining above a ratio of 1 for 7 days. For two horses with extended sampling, the plasma ratio remained above one for approximately 28 days after cobalt chloride administration. The effect of the administration of the vitamin B12 /cobalt supplement on the urine ratio was transient and reached a peak value of 10 which then rapidly declined. However, a urine ratio of 10 was exceeded, with the lower 95% confidence interval remaining above a ratio of 10 for 7 days after cobalt chloride administration. For the two horses with extended sampling, the urine ratio remained above 10 for about 18 days (442 h) after cobalt chloride administration even though the absolute cobalt urine concentration had dropped below the international threshold of 100 ng/mL after 96 h. Only one vitamin B12 /cobalt product was evaluated, a limited number of horses were included, the horses were not in full race training and the results may be specific to this population of horses. The results provide the basis for a potential strategy for allowing supplementation with vitamin B12 products, while controlling the misuse of high doses of cobalt, through a combination of international thresholds and ratios of cobalt to vitamin B12 , in plasma and urine.

NRF2 overexpression in mesenchymal stem cells induces stem-cell marker expression and enhances osteoblastic differentiation

Hypoxic environment has been suggested in stem cell culturing as their physiologic niche requires oxygen tension to maintain stemness. The administration of cobalt chloride (CoCl2) was widely applied in mimicking hypoxia for its economic advantages and convenience. We confirmed that CoCl2 could maintain stemness and promote the osteogenesis capacity of MSCs. However, CoCl2 could induce the apoptosis and hinder the proliferation of MSCs. To find out a potential method maintaining their stemness without threatening their survival, we analyzed the database of Gene Expression Omnibus (GEO) and proposed that NRF2 (nuclear factor erythroid-derived 2-like 2) might be the potential target. We found that knocking down NRF2 expression in MSCs impaired the expression of stem cell markers and the osteogenesis process even under hypoxic environment, but with NRF2 overexpression, the proliferation of MSCs was increased with significantly reduced rate of apoptosis. Therefore, our findings suggested that overexpressing NRF2 could be a potential method for maintaining stemness and preventing apoptosis in MSCs under oxidative stress.

Interlaboratory trial for the measurement of total cobalt in equine urine and plasma by ICP‐MS

Cobalt is an essential mineral micronutrient and is regularly present in equine nutritional and feed supplements. Therefore, cobalt is naturally present at low concentrations in biological samples. The administration of cobalt chloride is considered to be blood doping and is thus prohibited. To control the misuse of cobalt, it was mandatory to establish an international threshold for cobalt in plasma and/or in urine. To achieve this goal, an international collaboration, consisting of an interlaboratory comparison between 5 laboratories for the urine study and 8 laboratories for the plasma study, has been undertaken. Quantification of cobalt in the biological samples was performed by inductively coupled plasma-mass spectrometry (ICP-MS). Ring tests were based on the analysis of 5 urine samples supplemented at concentrations ranging from 5 up to 500ng/mL and 5 plasma samples spiked at concentrations ranging from 0.5 up to 25ng/mL. The results obtained from the different laboratories were collected, compiled, and compared to assess the reproducibility and robustness of cobalt quantification measurements. The statistical approach for the ring test for total cobalt in urine was based on the determination of percentage deviations from the calculated means, while robust statistics based on the calculated median were applied to the ring test for total cobalt in plasma. The inter-laboratory comparisons in urine and in plasma were successful so that 97.6% of the urine samples and 97.5% of the plasma samples gave satisfactory results. Threshold values for cobalt in plasma and urine were established from data only obtained by laboratories involved in the ring test. Copyright © 2017 John Wiley & Sons, Ltd.

Protective effect of grapes polyphenol concentrate “Fenokor” in terms of hypoxic myocardial injury

Polyphenols of grapes and their beneficial effects on human health have been known for a long time and still attract more and more research interest. The aim of the research was to reveal cardioprotective properties of polyphenols contained in grape concentrate “Fenokor” in terms of experimental histotoxic hypoxia. Materials and methods. The study was conducted on 21 adult male Wistar rats divided into 3 groups, 2 of which were administered CoCl2 aqueous solution in a dose of 60 mg/kg for 7 days intragastrically. The control group consisted of 5 intact animals. The rats of the second group (n = 8) after the introduction of the cobalt did not receive any treatment, the animals of the third group (n = 8) after the administration of cobalt chloride intragastrically, were given Fenokor aqueous solution at a dose of 2,5 ml/kg along with 0,05 ml of water orally. Morphological study was performed using light and electron microscopy. During the experiment the following biochemical parameters such as contents of malondialdehyde, oxidative modification of proteins were estimated. The results. The result of the influence of cobalt on the heart of animals in experiments is the development of severe cardiomyopathy that requires cardio-protection. Histological structure of myocardium observed in the second group of male rats after cobalt intoxication on the background of grape polyphenol concentrate generally reflected a tendency to minimize the damage extent which was manifested in the form of normalization of cell structures and muscle fibers. Application of Fenokor has demonstrated its antioxidant and cytoprotective properties, which contributed to myocardial structure preservation in rats exposed to histotoxic hypoxia.

CARDIOPROTECTIVE EFFECT OF NATIVE ANTIHYPOXANTS IN EXPERIMENTAL COBALT CARDIOMYOPATHY

The aim of research – the study of cardioprotective properties of antioxidants in terms of histotoxic hypoxia under experimental conditions. Materials and methods. The study was conducted on 20 adult male Wistar rats divided into 3 experimental groups, which for 7 days were intraperitoneally injected aqueous CoCl2 solution at a dose of 60 mg/kg. Rats of the first experimental group (n = 6) had no administered drug correction, a the second group of animals (n = 7) after the cobalt chloride daily injections was administered intragastrically Enoant Premium aqueous solution at a dose of 2.5 ml / kg, along with 0.05 ml of water, the rats the third test group (n = 7) after the administration of cobalt chloride were exposed to cytoflavin correction concentrate and grape polyphenols administered simultaneously. Studies of myocardium were conducted using light and electron microscopy. Results of the research. The result of the cobalt toxic effect on the heart of animals in experiments lead to the development of cardiomyopathy, which required timely cardioprotection. Morphological changes in the second group of rats, despite a slight improvement compared with the group without correction,were characterized, above all, by uneven from mild to severe edema of the myocardium. Structure of myocardium observed in the third group of male rats after cobalt intoxication, generally reflected a tendency to minimization of the extent of the damage, which was manifested in the form of normalization of cell structures and muscle fibers. Conclusion. The administration of succinic acid derivatives combined with the grape polyphenols demonstrated vivid cytoprotective properties evidenced by mostly preserved myocardium structure in rats exposed to histotoxic hypoxia in comparison to only administration of plant polyphenols group.

Inhibition of Prolyl Hydroxylase Domain-Containing Protein Downregulates Vascular Angiotensin II Type 1 Receptor

Inhibition of prolyl hydroxylase domain-containing protein (PHD) by hypoxia stabilizes hypoxia-inducible factor 1 and increases the expression of target genes, such as vascular endothelial growth factor. Although the systemic renin-angiotensin system is activated by hypoxia, the role of PHD in the regulation of the renin-angiotensin system remains unknown. We examined the effect of PHD inhibition on the expression of angiotensin II type 1 receptor (AT(1)R). Hypoxia, cobalt chloride, and dimethyloxalylglycine, all known to inhibit PHD, reduced AT(1)R expression in vascular smooth muscle cells. Knockdown of PHD2, a major isoform of PHDs, by RNA interference also reduced AT(1)R expression. Cobalt chloride diminished angiotensin II-induced extracellular signal-regulated kinase phosphorylation. Cobalt chloride decreased AT(1)R mRNA through transcriptional and posttranscriptional mechanisms. Oral administration of cobalt chloride (14 mg/kg per day) to C57BL/6J mice receiving angiotensin II infusion (490 ng/kg per minute) for 4 weeks significantly attenuated perivascular fibrosis of the coronary arteries without affecting blood pressure level. These data suggest that PHD inhibition may be beneficial for the treatment of cardiovascular diseases by inhibiting renin-angiotensin system via AT(1)R downregulation.

Different role of the ventral medial prefrontal cortex on modulation of innate and associative learned fear

Reversible inactivation of the ventral portion of medial prefrontal cortex (vMPFC) of the rat brain has been shown to induce anxiolytic-like effects in animal models based on associative learning. The role of this brain region in situations involving innate fear, however, is still poorly understood, with several contradictory results in the literature. The objective of the present work was to verify in male Wistar rats the effects of vMPFC administration of cobalt chloride (CoCl 2), a selective inhibitor of synaptic activity, in rats submitted to two models based on innate fear, the elevated plus-maze (EPM) and light-dark box (LDB), comparing the results with those obtained in two models involving associative learning, the contextual fear conditioning (CFC) and Vogel conflict (VCT) tests. The results showed that, whereas CoCl 2 induced anxiolytic-like effects in the CFC and VCT tests, it enhanced anxiety in rats submitted to the EPM and LDB. Together these results indicate that the vMPFC plays an important but complex role in the modulation of defensive-related behaviors, which seems to depend on the nature of the anxiety/fear inducing stimuli.

Lipid peroxidation, activity of Na+,K+-ATPase and exzymes of antioxidant defence in rats with nephropathy induced by cobalt chloride

Chronic parenteral administration of cobalt chloride (6 mg/kg) to male rats for 2 weeks or 1 month was accompanied by activation of lipid peroxidation (LPO), a decrease of superoxide dismutase activity and an increase of catalase activity. The membrane toxic action also resulted in a decrease of cortical and medullar Na+,K(+)-ATPase activity of kidneys, and the decrease in renal functions (glomerular filtration, renal water reabsorption, spontaneous diuresis, electrolyte excretion).

Cobalt Supplementation Promotes Hypoxic Tolerance and Facilitates Acclimatization to Hypobaric Hypoxia in Rat Brain

In the present study, we report the molecular mechanisms of action by cobalt in facilitating acclimatization to hypobaric hypoxia using male Sprague-Dawley rats as the model system. We determined hypoxic gasping time and survival time as a measure to assess the degree of tolerance of animals to hypobaric hypoxia by exposing the animals to an altitude of 10,668 m. Oral administration of cobalt chloride (12.5 mg Co/kg body weight, BW, for 7 days) increased gasping time and hypoxic survival time by 3 to 4 times compared to the control animals. This could be attributed to an increased expression and the DNA binding activity of hypoxia inducible transcriptional factor (HIF-1alpha) and its regulated genes, that is, erythropoietin (EPO), vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1), and nitric oxide synthase (NOS) levels. This in turn leads to better oxygenation, oxygen delivery, glucose transport, and maintenance of vascular tone, respectively, under oxygen-limited conditions. This was further confirmed by lower levels of lactate dehydrogenase (LDH) activity and lactate in the brain of cobalt + hypoxia group compared with animals exposed to hypoxia. Glucose levels also increased after cobalt supplementation. The findings of the study provide a basis for the possible use of cobalt for facilitating acclimatization to hypoxia and other conditions involving oxygen deprivation.

Effects of Hexaammine Cobalt (III) Chloride on Oxidative Stress-Related Parameters and Drug Metabolizing Enzymes in Mice

Hexaammine cobalt (III) chloride has been advocated as a potential anticarcinogenic compound. There is no information on the effects of this compound on oxidative stress-related parameters in animals. In the present study the effects of administration of hexaammine cobalt (III) chloride in drinking water to balb/c male mice at doses of 25, 50, and 100 ppm for 14 weeks were examined. The tissue distribution of the compound was seen in liver, kidney, lung, intestine, blood, and spleen. The effects of the compound were monitored on levels of lipid peroxidation, GSH content, and activities of SOD, catalase, GST, and Cyt P450, along with the liver and kidney function tests. The results show that the cobalt accumulated maximally in kidney followed by liver, intestine, blood, spleen, and lung in decreasing order, in a dose-dependent manner. GSH and GST also showed increase in a dose-dependent manner while SOD and catalase showed increase with the highest dose only. Liver and kidney function tests showed no untoward change with any dose at the end of the study. The results suggest an antioxidant potentiating effect of the hexaammine cobalt (III) chloride besides nontoxicity to liver and kidney. Since the ability to induce an increase of GSH and GST along with other detoxifying enzymes by anticarcinogenic agents has been reported to correlate with the inhibition of tumorigenesis, the cobalt complex might qualify as a potential cancer chemopreventive agent.

Induction of renoprotective gene expression by cobalt ameliorates ischemic injury of the kidney in rats.

Hypoxia in the tubulointerstitium has been thought to play pivotal roles in the pathophysiology of acute renal failure and the progression of chronic kidney disease. Pre-induction of hypoxia-inducible and renoprotective gene expression may protect subsequent ischemic injury. This study evaluated the efficacy of cobalt, which inhibits HIF-1 degradation and increases the expression level of hypoxia-related genes, in an acute ischemic tubulointerstitial injury model of rats. Ischemic renal injury was induced by 45-min clamping of renal pedicles with contralateral nephrectomy. Elevation of serum creatinine and morphologic injury after the ischemic insult was observed. Administration of cobalt chloride afforded striking functional improvement (mean +/- SEM creatinine in mg/dl: Co treatment group, 2.14 +/- 1.21; control, 3.69 +/- 1.43; P < 0.05) associated with amelioration of tubulointerstitial damage. Cobalt treatment also reduced macrophage infiltration significantly. In the kidney of rats treated with cobalt, mRNA levels of several genes that serve for tissue protection, such as HO-1, EPO, Glut-1, and VEGF, were increased before ischemic injury. Upregulation of HO-1 by cobalt was confirmed at the protein level. Subcutaneous injection of cobalt also ameliorated ischemic injury, which was associated with upregulation of renal HIF-1alpha protein expression. These results suggest that protection against hypoxic tubulointerstitial injury by cobalt administration is mediated by induction of renoprotective gene expression. HIF induction is one possible and attractive explanation for the observed effects.

Enhanced heme oxygenase activity increases the antioxidant defense capacity of guinea pig liver upon acute cobalt chloride loading: comparison with rat liver

Changes in the activity of so-called oxidative stress defensive enzymes, superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and heme oxygenase, as well as changes in lipid peroxidation and reduced glutathione levels, were measured in guinea pig and rat liver after acute cobalt loading. Cobalt chloride administration produced a much higher degree of lipid peroxidation in guinea pig than in rat liver compared with the control animals. The intrahepatic reduced glutathione content in control guinea pig was higher than that in rat, but was equally decreased in both species after cobalt administration. The enzymatic scavengers of free radicals, superoxide dismutase, catalase and glutathione peroxidase, were significantly decreased in rat liver after acute cobalt loading, and as a compensatory reaction, the heme oxygenase activity was increased (seven-fold). In guinea pig liver, only superoxide dismutase activity was depleted in response to cobalt-induced oxidative stress, while catalase and glutathione peroxidase were highly activated and the heme oxygenase activity was dramatically increased (13-fold). It is assumed that enhanced heme oxygenase activity may have important antioxidant significance by increasing the liver oxidative-stress defense capacity.

Heme oxygenase is the main protective enzyme in rat liver upon 6-day administration of cobalt chloride.

Changes in the activities of rat liver heme oxygenase (HO), superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR), as well as changes in lipid peroxidation and reduced glutathione (GSH) levels were measured after acute loading and chronic administration of cobalt chloride (CoCl2). Acute loading was achieved by a single subcutaneous injection of 60 mg CoCl2/kg body weight for 24 h. Chronic administration was performed by giving the same total amount of CoCl2 in small doses over longer periods of time: 30 mg CoCl2/kg daily for 2 days, 15 mg CoCl2/kg daily for 4 days, or 10 mg CoCl2/kg daily for 6 days. The results showed that HO activity was increased both after acute loading (7-fold increase) and upon 6-day administration of CoCl2 (5-fold increase). The GSH level, 24 h after a single injection of CoCl2, was lower than that of the control animals. However, upon chronic administration of small doses CoCl2, the level of GSH increased and was accompanied by an increase in GR activity. Chronic administration of CoCl2 produced persistent oxidative stress, which was illustrated with a continuous increase in lipid peroxidation. At the same time, under these conditions, the activities of oxidative-stress-protective enzymes were either inhibited (SOD, catalase) or not significantly changed (GPx). Collectively, these findings suggest that the sustained up-regulation of HO activity in rat liver upon 6 day administration of CoCl2 would be beneficial by providing the cells with antioxidants, biliverdin and bilirubin, and together with the increased levels of GSH would act as a part of the defence mechanisms against the cobalt-induced oxidative stress.

Calcium ions positively modulate follicle-stimulating hormone- and exogenous cyclic 3',5'-adenosine monophosphate-driven transcription of the P450(scc) gene in porcine granulosa cells.

Given the evident modulation of FSH-induced steroidogenesis by Ca2+ in granulosa cells, we here test the hypothesis that Ca2+ controls expression of the enzymatically rate-limiting cytochrome P450(scc) (CYP11A) gene. To test this postulate, we quantitated the ability of Ca2+ to regulate: 1) transcriptional activity of a transiently transfected luciferase reporter gene driven by a 2.32-kb 5'-upstream fragment of the porcine P450(scc) gene promoter region; and 2) accumulation of endogenous P450(scc) transcripts in primary monolayer cultures of porcine granulosa cells. To this end, granulosa cells were stimulated for 4 h with FSH (15 ng/ml, NIDDK-oFSH-20) or 8-Bromo-cAMP (8 Br-cAMP, 1 mM) in serum-free medium containing either 1.8 mM Ca2- or no added Ca2+ with 100 microM EGTA or 100 microM CoCl2. In the presence of extracellular Ca2+, FSH and 8 Br-cAMP stimulated expression of the transfected P450(scc) promoter-reporter fusion construct by 5.6 +/- 1.1 and 3.6 +/- 0.67-fold, respectively over Ca2+-containing unstimulated control (P < or = 0.04, n = 5-6 experiments). The foregoing two agonists augmented 4-h progesterone production by cultured granulosa cells by 1.8 +/- 0.11 and 1.6 +/- 0.16-fold, respectively (P < or = 0.001 for FSH and P < or = 0.01 for 8 Br-cAMP). FSH and 8 Br-cAMP also significantly elevated endogenous P450(scc) transcript levels as measured by homologous solution-hybridization RNase protection assay; i.e. by 3.1 +/- 0.49 and 2.9 +/- 0.45-fold, respectively (P < or = 0.001). In Ca2+-free/EGTA-supplemented medium, basal luciferase reporter-gene activity and endogenous P450(scc) messenger RNA accumulation in granulosa cells declined to 34 +/- 12% and 78 +/- 12%, respectively, of corresponding values in control (unstimulated Ca2+-containing) cultures. Extracellular Ca2+ deprivation inhibited the stimulatory effect of FSH (and 8 Br-cAMP) on P450(scc) promoter-luciferase reporter expression to 58 +/- 30% (and 58 +/- 23%), and restrained endogenous P450(scc) message accumulation to 86 +/- 15% (and 96 +/- 18%) of the value in Ca2+-containing control. Extracellular Ca2+ withdrawal suppressed FSH (and 8 Br-cAMP)-driven progesterone production over 4 h to basal levels but did not alter FSH-stimulated cAMP accumulation by granulosa cells. Ca2+-deprived cells exposed to serum-containing media regained P450(scc) responsiveness to both agonists. Antagonism of cellular uptake of Ca2+ and other divalent cations via administration of cobalt chloride (100 microM) inhibited FSH and 8 Br-cAMP's stimulation of endogenous (but not exogenous promoter-driven) P450(scc) gene expression. In contrast, granulosa-cell concentrations of messenger RNA's encoding sterol-carrier protein-2 (SCP-2) and the low density lipoprotein receptor were not altered by Ca2+ withdrawal. In summary, uptake of extracellular Ca2+ by porcine granulosa cells significantly potentiates transactivation of the endogenously expressed and exogenously transfected P450(scc) gene by FSH and 8 Br-cAMP. The agonistic impact of Ca2+ on P450(scc) promoter activity is requisite downstream of FSH-induced cAMP second-messenger signaling.

Impairment of Cardiac Energy Metabolism In Vivo Causes Hemodynamic Abnormality and Increases Cardiac Expression of Preproendothelin-1 mRNA

We investigated whether impairment of myocardial energy metabolism attenuates cardiac function and increases cardiac endothelin-1 (ET-1) gene expression in rats. Three weeks after commencing administration of cobalt chloride (CoCl2), an inhibitor of mitochondrial function, the peak positive first derivative of left ventricular (LV) pressure, an indicator of myocardial contractility, was significantly decreased in the CoCl2-treated rats. LV end-diastolic pressure and right ventricular systolic pressure were increased in the CoCl2-treated rats. Echocardiography showed that fractional shortening was significantly decreased in the CoCl2-treated rats. Myocardial expressions of acyl-CoA synthase mRNA, an enzyme involved in fatty acid utilization, was markedly decreased in the CoCl2-treated rats. Under such conditions, myocardial expression of preproendothelin-1 mRNA and atrial natriuretic peptide (ANP) mRNA, molecular markers of heart failure, was markedly increased in the CoCl2 rats. In conclusion, the data suggest that impairment of myocardial energy metabolism causes hemodynamic abnormality and increases molecular markers of heart failure (ET-1, ANP mRNA). These data suggest that myocardial energy metabolism is one of the factors involved in the upregulation of ET-1 gene expression in the failing heart.