Jan Magdalan

The protective effect of the Cornus mas fruits (cornelian cherry) on hypertriglyceridemia and atherosclerosis through PPARα activation in hypercholesterolemic rabbits.

Cornelian cherry (Cornus mas L.) fruits have been used in traditional cuisine and in folk medicine in various countries. This study was conducted to evaluate the constituents and impact of cornelian cherry (C. mas L.) fruits lyophilisate on lipid levels, PPARα protein expression, atheromatous changes in the aorta, oxido-redox state, and proinflammatory cytokines in hypercholesterolemic rabbits. The HPLC-MS method was used for determining active constituents in cornelian cherry. In a subsequent in vivo study the protective effect of the cornelian cherry on diet-induced hyperlipidemia was studied using a rabbit model fed 1% cholesterol. Cornelian cherry (100mg/kg b.w.) or simvastatin (5mg/kg b.w.) were administered orally for 60 days. Two iridoids - loganic acid and cornuside - and five anthocyanins were identified as the main constituents of the cornelian cherry. The administering of the cornelian cherry led to a 44% significant decrease in serum triglyceride levels, as well as prevented development of atheromatous changes in the thoracic aorta. Cornelian cherry significantly increased PPARα protein expression in the liver, indicating that its hypolipidemic effect may stem from enhanced fatty acid catabolism. Simvastatin treatment did not affect PPAR-α expression. Moreover, the cornelian cherry had a significant protective effect on diet-induced oxidative stress in the liver, as well as restored upregulated proinflammatory cytokines serum levels. In conclusion, we have shown loganic acid to be the main iridoid constituent in the European cultivar of the cornelian cherry, and proven that the cornelian cherry could have protective effects on diet-induced hypertriglicerydemia and atherosclerosis through enhanced PPARα protein expression and via regulating oxidative stress and inflammation.

alpha-Amanitin induced apoptosis in primary cultured dog hepatocytes.

Amatoxin poisoning is caused by mushroom species belonging to the genera Amanita, Galerina and Lepiota with the majority of lethal mushroom exposures attributable to Amanita phalloides. High mortality rate in intoxications with these mushrooms is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amatoxins. A wide variety of amatoxins have been isolated; however, alpha-amanitin (alpha-AMA) appears to be the primary toxin. Studies in vitro and in vivo suggest that alpha-AMA does not only cause hepatocyte necrosis, but also may lead to apoptotic cell death. The objective of this study was to evaluate the complex hepatocyte apoptosis in alpha-AMA cytotoxicity. All experiments were performed on primary cultured canine hepatocytes. The cells were incubated for 12 h with alpha-AMA at a final concentration of 1, 5, 10 and 20 microM. Viability test (MTT assay), apoptosis evaluation (TUNEL reaction, detection of DNA laddering and electron microscopy) were performed at 6 and 12 h of exposure to alpha-AMA. There was a clear correlation between hepatocyte viability, concentration of alpha-AMA and time of exposure to this toxin. The decline in cultured dog hepatocyte viability during the exposure to alpha-AMA is most likely preceded by enhanced cellular apoptosis. Our results demonstrate that apoptosis might contribute to pathogenesis of the severe liver injury in the course of amanitin intoxication, particularly during the early phase of poisoning.

Benzylpenicillin, acetylcysteine and silibinin as antidotes in human hepatocytes intoxicated with α-amanitin

Fatalities due to mushroom poisonings are increasing worldwide, with high mortality rate resulting from ingestion of amanitin-producing species. Intoxications caused by amanitin-containing mushrooms represent an unresolved problem in clinical toxicology since no specific and fully efficient antidote is available. The objective of this study was a comparative evaluation of benzylpenicillin (BPCN), acetylcysteine (ACC) and silibinin (SIL) as an antidotes in human hepatocytes intoxicated with alpha-amanitin (alpha-AMA). All experiments were performed on cultured human hepatocytes. Cytotoxicity evaluation of cultured cells using MTT assay and measurement of lactate dehydrogenase (LDH) activity was performed at 12, 24 and 48h of exposure to alpha-AMA and/or antidotes. The significant decline of cell viability and significant increase of LDH activity were observed in all experimental hepatocyte cultures after 12, 24 and 36h exposure to alpha-AMA at concentration 2microM. Exposure of the cells to alpha-AMA resulted also in significant reduction of cell spreading and attachment. However, addition of tested antidotes to experimental cultures significantly stimulated cell proliferation and attachment. In cell cultures exposed simultaneously to alpha-AMA and tested antidotes cytotoxic parameters (MTT and LDH) were not significantly different from control incidences. The cytoprotective effect of all antidotes was not dose-related, which reflects a high efficacy of all these substances. Administration of studied antidotes was not associated with any adverse effects in hepatocytes. The administration of ACC, BPCN or SIL to human hepatocyte cultures showed a similar strong protective effect against cell damage in alpha-AMA toxicity.

Early morphological and functional alterations in canine hepatocytes due to α-amanitin, a major toxin of Amanita phalloides.

The toadstool death cap (Amanita phalloides) and its subspecies, destroying angel (A. virosa) and death angel (A. verna) are responsible for nearly 95% of all fatal mushroom poisonings. High mortality rate in A. phalloides intoxications is principally a result of the acute liver failure following significant hepatocyte damage due to hepatocellular uptake of amanitins, the major toxins of this mushroom. This study evaluated early morphological and functional alterations in hepatocytes exposed to different concentrations of α-amanitin (α-AMA). All experiments were performed on cultured canine hepatocytes since intoxicated with A. phalloides dogs have clinical course and pathological findings similar to those seen in humans. The overall functional integrity and viability of cultured hepatocytes were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and by measurements of lactate dehydrogenase (LDH), total protein, and urea levels. Our results showed that the course of α-AMA toxicity in cultured dog hepatocytes is divided into two phases. The first phase comprises functional cell impairments expressed by significant increase of LDH activity and inhibition of protein and urea synthesis when compared with the control group. This is followed by discrete changes in hepatocyte ultrastructure, including marginalization and condensation of nuclear chromatin, as well as formation of the foamlike cytoplasm. The second stage is lethal and is characterized by ongoing necrosis, and/or apoptosis. This may be related to dose of toxin and time of exposure.

Influence of commonly used clinical antidotes on antioxidant systems in human hepatocyte culture intoxicated with α-amanitin

α-Amanitin (α-AMA) is the main toxin of Amanita phalloides and its subspecies (A. virosa and A. verna). The primary mechanism of α-AMA toxicity is associated with protein synthesis blocking in hepatocytes. Additionally, α-AMA exhibits prooxidant properties that may contribute to its severe hepatotoxicity. The aim of the present study was to assess the effect of α-AMA on lipid peroxidation and the activities of superoxide dismutase (SOD) and catalase (CAT) in human hepatocyte culture. The effects of benzylpenicillin (BPCN), N-acetyl-L-cysteine (ACC), and silibinin (SIL) on SOD and CAT activities and on lipid peroxidation in human hepatocyte culture intoxicated with α-AMA were also examined. In human hepatocyte culture, 48-hour exposure to α-AMA at a 2-μM concentration caused an increase in SOD activity, a reduction of CAT activity, and a significant increase in lipid peroxidation. Changes in SOD and CAT activity caused by α-AMA could probably enhance lipid peroxidation by increased generation of hydrogen peroxide combined with reduced detoxification of that oxygen radical. The addition of antidotes (ACC or SIL) to the culture medium provided more effective protection against lipid peroxidation in human hepatocytes intoxicated with α-AMA than the addition of BPCN, possessing no antioxidant properties.

Effects of morin-5′-sulfonic acid sodium salt (NaMSA) on cyclophosphamide-induced changes in oxido-redox state in rat liver and kidney

Cyclophosphamide (CPX) is an anticancer drug with immunosuppressive properties. Its adverse effects are partly connected to the induction of oxidative stress. Some studies indicate that water-soluble derivative of morin—morin-5′-sulfonic acid sodium salt (NaMSA) exhibits strong antioxidant activity. The aim of present study was to evaluate the effect of NaMSA on CPX-induced changes in oxido-redox state in rat. Experiment was carried out on Wistar rats divided in three experimental groups (N = 12) receiving: 0.9% saline, CPX (15 mg/kg) or CPX (15 mg/kg) + NaMSA (100 mg/kg), respectively, and were given intragastrically for 10 days. Malondialdehyde (MDA) and glutathione (GSH) concentrations and superoxide dismutase (SOD) activity were determined in liver and kidneys. Catalase (CAT) activity was assessed only in liver. Treatment with CPX resulted in significant decrease in MDA level in both tissues, which was completely reversed by NaMSA treatment only in liver. In comparison to the control group significant decrease in SOD activity were observed in both tissues of CPX receiving group. In kidneys this parameter was fully restored by NaMSA administration. CPX evoked significant decrease in GSH concentration in kidneys, which was completely reversed by NaMSA treatment. No significant changes were seen in GSH levels and CAT activity between all groups in liver. Results of our study suggest that CPX may exert significant impact on oxido-redox state in both organs. NaMSA fully reversed the CPX-induced changes, especially MDA level in liver, SOD activity and GSH concentration in kidneys and it may be done by enhancement of activity/concentration of endogenous antioxidants.

Benzylpenicyllin and acetylcysteine protection from α-amanitin-induced apoptosis in human hepatocyte cultures

High mortality rate in Amanita phalloides (death cap) intoxications is a result of the acute liver failure following hepatocyte damage due to hepatocellular uptake of amatoxins. α-Amanitin (α-AMA), the major amatoxin, blocks a RNA polymerase II, which results in inhibition of transcription of DNA and protein synthesis processes and leads to hepatocyte death. α-AMA is also a strong apoptosis inductor and may play a significant role in pathogenesis of hepatic damage in course of amanitin intoxication. The aim of this study was to examine mechanisms of α-AMA-induced apoptosis in human hepatocytes, as well as in determining if commonly clinically used antidotes benzylpenicillin (BPCN) and N-acetylcysteine (ACC) are able to protect human hepatocytes against α-AMA-induced apoptosis. The experiment was performed on cultured human hepatocytes. Viability of cultured hepatocytes was assessed using the MTT assay, whereas apoptosis processes were evaluated by the electron microscopy, detection of DNA laddering, determination of caspase-3 activity, and measuring annexin V, p53 and Bcl-2 protein concentration. Cytotoxicity and apoptosis evaluation were performed after 24 h of exposure to α-AMA and/or tested antidotes.Both ACC and BPCN were well tolerated by human hepatocyte cultures, and exposure to those substances did not reduce cell viability nor induce apoptosis. Exposure of hepatocytes to α-AMA at concentration 2μM resulted in derangement of cell cultures, apoptosis and significant reduction in cell viability. α-AMA-induced apoptosis in human heptocyte cultures is p53- and caspase-3-dependent. Human hepatocyte cultures are exposed simultaneously to α-AMA and tested antidotes (BPCN or ACC) showed significantly higher cell viability and significantly lower values of apoptosis markers compared to the cultures exposed to α-AMA only.

Effects of melatonin on lipid peroxidation and antioxidative enzyme activities in the liver, kidneys and brain of rats administered with benzo(a)pyrene.

Benzo(a)pyrene [B(a)P] is a widespread pollutant with a mutagenic, carcinogenic and strong prooxidative properties. The present study evaluated the melatonin effects on lipid peroxidation products levels and on activity of antioxidative enzymes in the course of B(a)P intoxication. Control rats were treated with 0.9% NaCl; another group was given 10mg melatonin/kg bw; a third group was injected twice a week with B(a)P at the dose of 10mg/kg bw; the fourth group received both B(a)P and melatonin at the dose as mentioned above. The experiment continued for 3 months. In homogenates of brain, liver and kidneys lipid peroxidation was appraised by evaluation of malonyldialdehyde and 4-hydroxyalkenal (MDA+4HDA) levels. Activities of glutathione peroxidase (GPx), superoxide dysmutase (SOD) and catalase (CAT) and concentration of reduced glutathione (GSH) were also estimated. In animals receiving both B(a)P and melatonin, lower levels of MDA+4HDA were observed in all organs as compared to the group treated with B(a)P only. Following administration of B(a)P, GSH level decreased in brain and kidney. Melatonin in combination with B(a)P induced rises in the GSH level in liver and brain, as compared to the receiving B(a)P alone. The activity of SOD increased in the rats treated with melatonin alone but the highest activity was observed in rats treated with B(a)P plus melatonin. CAT activity in the melatonin-treated group increased in brain and liver. Similar to SOD, activity of the enzyme significantly increased in the group treated in combination with B(a)P and melatonin, as compared to the remaining groups in all tested tissues. The results suggest that melatonin protects cells from the damaging action of B(a)P. According to our knowledge, there are no studies describing the effects of melatonin on lipid peroxidation markers and antioxidative enzymes during intoxication of B(a)P in the brain, liver and kidneys. The results of present study give a perspective for further studies of its free radical scavenger properties in prevention of oxidative stress dependent diseases, among others cancers caused by carcinogens such as B(a)P.

Vipera berus bites in the Region of Southwest Poland--a clinical analysis of 26 cases.

OBJECTIVE Vipera berus is the only naturally occurring venomous snake in Poland. Its venom is primarily vasculotoxic and evokes both local and systemic findings. The aim of the study was to review a series of clinical cases of V berus bites occurring in southwest Poland. METHODS The charts of 26 patients (age range, 16-66 years; mean, 42 years) hospitalized with V berus bites were retrospectively analyzed using a data collection tool. Demographic and clinical data were extracted. RESULTS The most common local findings of envenomation were edema of the bitten limb with associated extravasations observed in 24 (92.3%) patients, but in only 1 (3.8%) case did the edema spread to the trunk. In 22 (84.6%) cases edema disappeared within 2 weeks after the bite. Systemic disturbances observed in the patients were: shock (1 case), mild transient hypotension (1 case), prolonged hypotension (3 cases), bronchospasm and laryngeal edema (1 case), diarrhea (1 case), transient supraventricular arrhythmias (2 cases), neutrophilic hyperleukocytosis (2 cases), and thrombocytopenia below 50000 cells/microL (5 cases). In 16 patients (61.5%) the envenomation was classified as moderate and this type was predominant. Six cases were classified as severe. No fatal case was reported. Treatment included the administration of specific antivenom in 14 cases (in all severe and half of moderate cases) and symptomatic treatment applied in all cases. CONCLUSIONS Moderate envenomation prevailed among the patients analyzed in the study. Antivenom treatment is primarily necessary in cases of severe (grade 3) and in some cases of moderate (grade 2) envenomation, especially in patients with persistent or recurring hypotension.

Suicidal overdose with relapsing clomipramine concentrations due to a large gastric pharmacobezoar

The paper presents a case of fatal intoxication after massive sustained-release clomipramine overdosage with prolonged toxicity related to a large gastric pharmacobezoar. 42-year-old female was admitted to the toxicology unit 14 h after drugs ingestion. At admission patient was deeply unconscious, required controlled mechanical ventilation. Serum total level of TCAs was 1955 ng/mL. Gastric lavage revealed no pills. Within the next 12h the patients clinical condition improved. TCAs level decreased to 999 ng/mL. However, after another 10h the clinical condition started deteriorating again and the patient went into a deep coma requiring controlled mechanical ventilation. TCAs level increased to 2011 ng/mL. X-ray and computed tomography revealed large pharmacobezoar consisted from radio-opaque pills. In the 28th h of hospitalization gastrotomy was performed, confirming presence of pharmacobezoar formed from Anafranil SR tablets. After surgery TCAs level was gradually decreasing. However, the patients condition did not improve, she died 32 h after gastrotomy. Post-mortem analyses revealed drug and its metabolite toxic levels in blood (clomipramine - 1729 ng/mL, norclomipramine - 431 ng/mL) and toxic levels in internal organs: myocardium (clomipramine - 14,420 ng/g, norclomipramine - 35,930 ng/g), vitreous humor (clomipramine - 1000 ng/mL, norclomipramine - 3110 ng/mL). Described case report indicates that sustained release clomipramine tablets may form pharmacobezoar. X-ray and computed tomography examinations should be considered in cases of massive abuse of sustained release clomipramine, particularly if symptoms of intoxication are recurrent or persistent.