Biodistribution of size-selected lyophilisomes in mice

Lyophilisomes are a novel class of proteinaceous biodegradable nano/microparticle capsules developed for tumor drug delivery. The in vivo characteristics of lyophilisomes are unknown and, therefore, the time course of biodistribution of sized albumin-based lyophilisomes in CD1 mice after intravenous administration was studied. Lyophilisomes, prepared from Dylight680-labeled albumin, were sized using a sucrose gradient centrifugation methodology and four fractions with a mean size of approximately 200 nm, 400 nm, 550 nm, and 650 nm were pooled for in/ex vivo localization, (immuno)histochemistry and biochemical analysis. Lyophilisomes were rapidly taken out of the circulation by the liver and spleen. Immunohistochemistry revealed that lyophilisomes were taken up in the liver by F4/80 positive macrophages, and in the spleen by Sign-R1 positive macrophages specifically located in the marginal zones. Lyophilisomes were most likely degraded by the liver and spleen and subsequently excreted via the urine, as high levels of degraded Dylight680-labeled albumin were detected in the urine. This was corroborated by electron microscopy of the spleen, which showed intact lyophilisomes in the marginal zone 5 and 30 min after injection, but not after 2 h. In conclusion, IV injected lyophilisomes are rapidly entrapped by liver and splenic macrophages, biodegraded, and excreted in the urine.     

Short- and long-term distribution and toxicity of gold nanoparticles in the rat after a single-dose intravenous administration

Surface chemistry plays an important role in gold nanoparticles (AuNPs) stability and biocompatibility, which are crucial for their implementation into the clinical setting. We evaluated short- (30 min) and long-term (28 days) biodistribution and toxicity of ~ 20 nm citrate- and pentapeptide CALNN-coated AuNPs after a single intravenous injection in rats. The pattern of AuNPs distribution in Cit- and CALNN-AuNPs-injected rats was very similar in the assessed time-points. Both AuNPs were quickly removed from the bloodstream and preferentially accumulated in the liver. At 28 days liver remained the main accumulation site but at significantly lower levels compared to those found at 30 min. Spleen atrophy and hematological findings compatible with mild anemia were observed in CALNN-AuNPs-administered rats. Under our experimental conditions, surface coating had more impact on toxicity rather than on biodistribution of the AuNPs. Improvements in the design of capping peptides need to be done to increase biomedical applicability of peptide-coated AuNPs.From the Clinical EditorThe biodistribution and toxicity of ~ 20 nm citrate- and pentapeptide CALNN-coated gold nanoparticles was investigated after a single intravenous injection in rats. Rapid clearance and hepatic accumulation was found at 30-minutes, whereas mild anemia and spleen atrophy was seen 28 days post injection. The authors also concluded that the toxicity was related to the capping proteins as opposed to the biodistribution of the particles, providing important suggestion for future design of gold nanoparticles.     

Electron microscopic ultrastructural study on the toxicological effects of AgNPs on the liver,kidney and spleen tissues of albino mice

The present study deals with the intraperitoneal administration of 500, 1000, 3000, and 5000 mg/kg of AgNPs in albino mice for 28 days to evaluate the potential toxicological effects of AgNPs on blood biochemical parameters and to investigate the light and electron microscopic histopathological alterations on three major targets organs i.e., liver, kidney and spleen. The AgNPs was well tolerated and no mortality was observed even at the highest dose i.e., 5000 mg/kg. Mice treated with 500 and 1000 mg/kg AgNPs did not show significant behavioral, biochemical and ultrastructural pathological changes. Mice treated with 1000 mg/kg AgNPs produces little ultrastructural alteration in liver, kidney and spleen. However, mice treated with 3000 and 5000 mg/kg AgNPs revealed significant changes in biochemical parameters. Electron microscopic ultrastructural investigation of liver and kidney shows that the administration of 3000 and 5000 mg/kg AgNPs revealed irregularity in the nuclear membrane, nuclear chromatin condensations, degenerated hepatocytes, swollen and pleomorphic mitochondria with distorted cristae, extensive dilation of rough endoplasmic reticulum, destructed cytoplasm, hypertrophied and fused podocytes and thickened basement membrane in the endothelial cells of the proximal tubules. The spleen sections at 3000 and 5000 mg/kg AgNPs revealed megakaryocytes hyperplasia, lobulations, invaginations and folding of nuclei and nuclear membrane. The present research indicates that AgNPs were well tolerated at the lower doses, but significant alterations in liver, kidney and spleen were observed at the higher doses tested. It is, therefore, suggested that further studies are needed for the minimization of the observed side effects, especially at higher doses before AgNPs being applied in pharmaceutical application.     

Lead accumulation and metallothionein content in female rats of different ages and generations after daily intake of Pb-contaminated food

Female Wistar rats of different ages (45, 90 and 140 days) and generations (mothers and offspring) were fed a feed containing 2.0 mg of Pb kg⁻¹ daily from weaning and the Pb accumulation was determined in different organs and in maternal milk, in addition metallothioneins (MTs) content was determined in the liver and kidneys. The results showed that Pb accumulation exhibited the following pattern: bone > liver > kidney > gut > blood cells > muscle > brain > ovary. Bones accumulated the most Pb in all animals, with its concentration increasing with age and prenatal exposure. Pb accumulation in the liver, kidney and blood cells, did not follow a consistent pattern with increasing age and our data did not indicate a relationship between the presence of MTs in liver and kidney and metal accumulation in these organs. However, in the offspring and with increasing age, Pb accumulated in more organs. Mothers fed with Pb produced contaminated milk, exposing their offspring to the metal via nursing Thus, increasing age and prenatal exposure increases susceptibility to Pb toxicity-induced damage.     

Efficacy of voriconazole in a murine model of invasive paecilomycosis

We studied the efficacy of voriconazole (VRC) and amphotericin B (AMB) in an immunosuppressed murine model of disseminated infection by two strains of Paecilomyces lilacinus. Mice were treated with VRC 60 mg/kg/day orally or AMB 3 mg/kg/day intraperitoneally, beginning 1 day after infection and continuing for 9 days. To avoid rapid clearance of VRC, animals receiving VRC and the control group were given grapefruit juice instead of water. VRC significantly prolonged survival with respect to the group treated with AMB and the control group for both strains (P = 0.005 and P < 0.0001, respectively, for strain FMR 5522; and P = 0.0002 and P < 0.0001, respectively, for strain FMR 8252). VRC reduced the fungal load in the spleen, kidneys and liver of infected mice for both strains tested. Survival of mice challenged with strain FMR 8252 treated with AMB did not differ from that of the control group (P = 0.223), being worse than that of the mice treated with VRC (P = 0.0002). AMB was not able to reduce the tissue burden in any organ with respect to the control group for both strains studied.     

Subchronic,reproductive, and maternal toxicity studies with tertiary butyl acetate (TBAC)

Tertiary-butyl acetate (TBAC) was tested for subchronic toxicity in rats and mice and reproductive toxicity in rats at inhalation concentrations of 0, 100, 400 or 1600 ppm. An oral maternal toxicity study was conducted in rats at dose levels of 0, 400, 800, 1000 and 1600 mg kg⁻¹ d⁻¹. In the inhalation studies, hematology, clinical chemistry, urinalysis, gross pathology and the majority of body weight and feed consumption values were unaffected. Exposure to TBAC at concentrations of 400 ppm and higher caused transient hyperactivity in mice and some evidence of increased motor activity counts in male rats at the 1600 ppm exposure level. TBAC caused α₂u-globulin accumulation in male rat kidneys from all exposure groups and increased liver weights in 1600 ppm rats and mice. Levels of thyroxin were decreased in male mice exposed to 1600 ppm TBAC for 4 weeks but otherwise thyroid endpoints were unaffected in rats and mice at either the 4 or 13 weeks time points. There was no evidence or immunotoxicity or reproductive toxicity in rats. Pregnant rats receiving 1000 mg kg⁻¹ d⁻¹ TBAC exhibited severe signs of acute neurotoxicity and decreased feed consumption and body weight gain. Fetal viability and growth were unaffected.     

Biodistribution and biopersistence of ceria engineered nanomaterials: size dependence

The aims were to determine the biodistribution, translocation, and persistence of nanoceria in the brain and selected peripheral organs. Nanoceria is being studied as an anti-oxidant therapeutic. Five, 15, 30, or 55 nm ceria was iv infused into rats which were terminated 1, 20, or 720 h later. Cerium was determined in blood, brain, liver, and spleen. Liver and spleen contained a large percentage of the dose, from which there was no significant clearance over 720 h, associated with adverse changes. Very little nanoceria entered brain parenchyma. The results suggest brain delivery of nanoceria will be a challenge.From the Clinical EditorThis team of investigators revealed that nanoceria, which is being studied as an anti-oxidant, has very limited uptake by the brain regardless of the range of sizes studied, suggesting major challenges in the application of this novel approach in the central nervous system.     

Dietary exposure of juvenile female mice to polyhalogenated seafood contaminants (HBCD,BDE-47, PCB-153, TCDD): Comparative assessment of effects in potential target tissues

Fish represents source of nutrients and major dietary vehicle of lipophilic persistent contaminants. The study compared the effects of two legacy and two emerging fish pollutants (Hexabromocyclododecane HBCD; 2,2′,4,4′-Tetrabromodiphenyl ether BDE-47; 2,2′,4,4′,5,5′-Hexachlorobiphenyl PCB-153; 2,3,7,8-Tetrachlorodibenzo-p-doxin TCDD) in juvenile female mice exposed through a salmon based rodent diet for 28 days (dietary doses: HBCD 199 mg/kg bw/day; BDE-47 450 μg/kg bw/day; PCB-153 195 μg/kg bw/day; TCDD 90 ng/kg bw/day). Dose levels were comparable to previously reported developmental Lowest Observed Adverse Effect Levels. None of the treatments elicited signs of overt toxicity, but HBCD increased relative liver weight. All compounds caused changes in liver, thymus and thyroid; spleen was affected by BDE-47 and PCB-153; no effects were seen in uterus and adrenals. Strongest effects in thyroid follicles were elicited by PCB-153, in thymus and liver by BDE-47. HBCD and BDE-47 induced liver fatty changes, but appeared to be less potent in the other tissues. HBCD, BDE-47 and TCDD increased serum testosterone levels and the testosterone/estradiol ratio, suggesting a potential involvement of pathways related to sex steroid biosynthesis and/or metabolism. The results support the role of toxicological studies on juvenile rodents in the hazard characterization of chemicals, due to endocrine and/or immune effects.     

Marine actinobacterial mediated gold nanoparticles synthesis and their antimalarial activity

Streptomyces sp LK-3 (JF710608) mediated Gold nanoparticles (Au-N-LK3) were found within the size range of 5-50 nm. Au-N-LK3 treatment in Plasmodium berghei ANKA (PbA) infected mice delayed the parasitemia rise (~ 6%) compared to PbA infection on 8 days post infection. Survivability of mice increases to ~ 85% in Au-N-LK3 treated mice in contrast to in PbA (~ 50%) infected mice in 8 dpi with respect to control. During Au-N-LK3 treatment in PbA infection, histomorphological analysis revealed as such no change in spleen and liver tissue during 8 dpi. Our results confirmed up-regulation of TGF-β and down-regulation of TNF-α in tissue and serum level in PbA infected Au-N-LK3 treated mice compared to PbA infection. No significant changes were found in the hatchability of Artemia embryos upto 8 mg. The results obtained suggest that the Au-N-LK3 possess anti-malarial activity and could be considered as a potential source for anti-malarial drug development.From the Clinical EditorThese investigators present a method of marine actinobacteria mediated synthesis of gold nanoparticles, resulting in nanoparticles that possess anti-malarial activity and could be considered in future anti-malarial drug development.     

Effects of combined exposure to formaldehyde and benzene on immune cells in the blood and spleen in Balb/c mice

Formaldehyde and benzene are the two major indoor air pollutants due to their prevalence and toxicity. This study aimed to explore the toxic effect on the spleen and relevant immune responses of Balb/c mice caused by exposure to a combination of formaldehyde and benzene. Balb/c mice were divided randomly into five groups (n = 9/group): blank control group (Ctrl); solvent ([corn] Oil) control; formaldehyde only (FA, 3 mg/m³); benzene only (BZ, 150 mg/kg BW); and, formaldehyde + benzene group (FA + BZ). Exposures were performed for 8 h/day, 5 day/week, for 2 weeks. Tail blood was collected after the final exposure; 24-h later, the mice were euthanized to permit assessment of a variety of immune endpoints. The endpoints’ three areas were: (1) in living mice, body weight and delayed-type hypersensitivity (DTH) responses; (2) in blood, immune cell counts and serum antibody levels (serum hemagglutination); and, (3) in spleen samples, reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), caspase-3 (cell apoptosis) levels and lymphocyte proliferation. In this study we fund (1) BZ and FA + BZ exposure can lead to the reduction in the number of some immune cells in peripheral blood; (2) Formaldehyde has certain synergistic effects on benzene-induced cytotoxicity in peripheral blood, (3) FA, BZ and FA + BZ exposure can lead to ROS and GSH depletion in spleen cells, and spleen cell apoptosis (caspase-3 increased) may be one of the downstream events, decreased splenic lymphocyte proliferation; and (4) the FA + BZ combined exposure can lead to the decreased body weight, serum antibody level (by serum hemagglutination assay).     

Protective effect of a marine polyphenol,dieckol against carbon tetrachloride-induced acute liver damage in mouse

In this study, the hepatoprotective effect of dieckol on carbon tetrachloride (CCl₄) induced hepatic damages in ICR mice liver was investigated. Mice were randomly divided into 4 groups such as saline treated (negative control), CCl₄ treated (positive control), CCl₄ + dieckol (5 mg/kg mouse) and CCl₄ + dieckol (25 mg/kg mouse), respectively. The body weights and survival rates of mice, followed by dieckol treatments were significantly increased compared to the positive control. The level of GOT, GPT and MDA in the serum of the dieckol treated groups were reduced dose dependently than the control, significantly. The antioxidant enzymes including CAT, and GSH-px levels were increased significantly compared to the positive control. However, no significant differences were observed on hepatic histophathological analysis in dieckol treated groups dose dependently. Down-regulation of Bax and up-regulation of Bcl-xl protein expressions were observed in liver tissues of the dieckol administered groups. These results suggested that, dieckol can be developed as a therapeutic agent for liver disease by oxidative stress.     

Evaluation of propargyl alcohol toxicity and carcinogenicity in F344/N rats and B6C3F1/N mice following whole-body inhalation exposure

Propargyl alcohol (PA) is a high production volume chemical used in synthesis of many industrial chemicals and agricultural products. Despite the potential for prolonged or accidental exposure to PA in industrial settings, the toxicity potential of PA was not well characterized. To address the knowledge gaps relevant to the toxicity profile of PA, the National Toxicology Program (NTP) conducted 2-week, 14-week and 2-year studies in male and female F344/N rats and B6C3F1/N mice. For the 2-week inhalation study, the rats and mice were exposed to 0, 31.3, 62.5, 125, 250 or 500 ppm. Significant mortality was observed in both rats and mice exposed to ≥125 ppm of PA. The major target organ of toxicity in both mice and rats was the liver with exposure-related histopathological changes (250 and 500 ppm). Based on the decreased survival in the 2-week study, the rats and mice were exposed to 0, 4, 8, 16, 32 or 64 ppm of PA in the 14-week study. No treatment-related mortality was observed. Mean body weights of male (≥8 ppm) and female mice (32 and 64 ppm) were significantly decreased (7–16%). Histopathological changes were noted in the nasal cavity, and included suppurative inflammation, squamous metaplasia, hyaline droplet accumulation, olfactory epithelium atrophy, and necrosis. In the 2-year inhalation studies, the rats were exposed to 0, 16, 32 and 64 ppm of PA and the mice were exposed to 0, 8, 16 and 32 ppm of PA. Survival of male rats was significantly reduced (32 and 64 ppm). Mean body weights of 64 ppm male rats were significantly decreased relative to the controls. Both mice and rats showed a spectrum of non-neoplastic changes in the nose. Increased neoplastic incidences of nasal respiratory/transitional epithelial adenoma were observed in both rats and mice. The incidence of mononuclear cell leukemia was significantly increased in male rats and was considered to be treatment-related. In conclusion, the key findings from this study indicated that the nose was the primary target organ of toxicity for PA. Long term inhalation exposure to PA led to nonneoplastic changes in the nose, and increased incidences of respiratory/transitional epithelial adenomas in both mice and rats. Increased incidences of harderian gland adenoma may also have been related to exposure to PA in male mice.     

Acute and subacute toxicity studies on triptolide and triptolide-loaded polymeric micelles following intravenous administration in rodents

Except its anti-tumour effects, triptolide (TP) also shows multiple pharmacological side activities, such as immune-suppressive and male anti-fertility. To increase the therapeutic index of TP, a novel polymeric micelle system containing TP (TP-PM) has been developed to treat tumour. Our previous studies have demonstrated the good anti-tumour efficacy of TP-PM. This paper investigated the acute toxicity in mice and subacute toxicity in rats of TP-PM and TP. Results demonstrated that the LD₅₀ for TP-PM and TP administered intravenously were 1.06 mg/kg and 0.83 mg/kg in mice, respectively. In subacute toxicity study, TP-PM and TP were administered intravenously at the dose levels of 0.1 mg/kg and 0.3 mg/kg for 14 d. Compared to the control, there was significant decrease in the serum AST activities, the testis ACP activities, thymus index, testis index, and significant increase in spleen index, and obvious histopathological changes in rats treated with TP, however, the toxicities of TP-PM on liver, kidney, testis and spleen are slighter than TP. Compared to TP, TP-PM significantly increased the ACP activity of the testis and decreased the MDA level in serum. So, the polymeric micelles may be a novel drug delivery carrier of TP for reducing the toxicities of TP.     

Oxidized casein impairs antioxidant defense system and induces hepatic and renal injury in mice

ScopeOxidized protein products (OPPs) can be easily found in meat and milk during processing and storage. Evidence supports that accumulation of endogenous OPPs plays a negative role in physiological metabolism. However, the impacts of dietary OPPs and the mechanisms have not been elucidated yet. The present study evaluated whether oral oxidized casein would destruct the antioxidant defense system and cause potential oxidized injury in mice liver and kidney.Methods and resultsWe performed oxidized casein (modified respectively by H₂O₂–Cu and HClO) feeding experiments using KM mice (20–22 g). A 10-weeks feeding of oxidized casein as basal protein caused oxidative stress by increasing protein carbonylation (PC), advanced oxidation protein products (AOPPs), dityrosine (Dityr), lipid peroxidation and ROS levels in mice liver, kidney and blood (P < 0.05). In mice liver and kidney, the mRNA expression of Nrf2, γ-GCS, HO-1, GPX-3, and GPX-4 up-regulated, the protein level of Nrf2 in nucleus increased. However, activities of anti-oxidant enzymes (CAT, SOD, and GPX) decreased (P < 0.05). Moreover, histopathological examination displayed the formation of fibrous septa in mice liver and kidney after oxidized casein feeding.ConclusionOxidized casein impairs antioxidant defense system and induces hepatic and renal fibrosis.     

Nicotinamide N-methyltransferase (NNMT) and 1-methylnicotinamide (MNA) in experimental hepatitis induced by concanavalin A in the mouse

Nicotinamide N-methyltransferase (NNMT), which converts nicotinamide (NA) to 1-methylnicotinamide (MNA), is up-regulated in the cirrhotic liver. Because MNA displays PGI₂-dependent anti-inflammatory effects, the up-regulation of NNMT may play a regulatory role in liver inflammation. In the present work, we analyzed changes in NNMT activity in the liver and concomitant changes in the concentration of endogenous MNA in plasma in T-cell dependent hepatitis induced by concanavalin A (ConA) in BALB/c mice. Furthermore, we tested whether exogenous MNA possessed a protective effect against ConA-induced hepatitis. Development of liver injury induced by ConA (10 mg/kg, iv) was characterized by measurements of plasma concentration of alanine aminotransaminase (ALT), inflammatory cytokines (IFNγ and TNFα) and by histopathological examination. ConA-induced hepatitis was characterized by an early activation of inflammatory cytokines (IFNγ; from below 0.05 ng/ml to 23.72 ± 8.80 ng/ml; TNFα; from 0.07 ± 0.01 ng/ml to 0.71 ± 0.12 ng/ml, 2 h after ConA), an elevation of ALT (from 40.65 ±3.2 U/l to 5,092.20 ± 1,129.05 U/l, 8 h after ConA) and by morphological signs of severe liver inflammation and injury (24 h after ConA). In mice injected with ConA, NNMT activity in the liver was up-regulated approximately 2-fold to 3-fold, 8–24 h after ConAinjection. The concentration of MNA and its metabolites (Met-2PY and Met-4PY) in plasma were elevated approximately 2-fold 8 h after ConA injection. Exogenous MNA (100 mg/kg, iv) diminished ConA-induced liver injury, and this effect was reversed by an antagonist of the prostacyclin receptor, RO 3244794 (10 mg/kg,po). In conclusion, the present study demonstrated that hepatic NNMT activity and MNA concentration in plasma significantly increased during the progression of ConA-induced hepatitis in mice. This response may play a hepatoprotective role compatible with the PGI2-releasing properties of MNA.     

Cholecystokinin protects mouse liver against ischemia and reperfusion injury

BackgroundCholecystokinin (CCK), as a gastrointestinal hormone, has an important protective role against sepsis or LPS-induced endotoxic shock. We aim to address the role of CCK in hepatic ischemia followed by reperfusion (I/R) injury.Materials and methodsA murine model of 60 min partial hepatic ischemia followed by 6 h of reperfusion was used in this study. CCK and CCKAR Levels in blood and liver were detected at 3 h, 6 h, 12 h and 24 h after reperfusion. Then the mice were treated with CCK or proglumide, a nonspecific CCK-receptor (CCK-R) antagonist. Mice were randomly divided into four groups as follows: (1) sham group, in which mice underwent sham operation and received saline; (2) I/R group, in which mice were subjected to hepatic I/R and received saline; (3) CCK group, in which mice were subjected to hepatic I/R and treated with CCK (400 μg/kg); (4) proglumide group (Pro), in which mice underwent hepatic I/R and treated with proglumide (3 mg/kg); CCK and proglumide were administrated via tail vein at the moment of reperfusion. Serum AST (sAST) and serum ALT (sALT) were determined with a biochemical assay and histological analysis were performed with hematoxylin-eosin (H&E). Cytokines (IL-1β, IL-6, IL-10, TNF-α) expressions in blood were determined with enzyme-linked immunosorbent assay (ELISA). The MPO (myeloperoxidase) assay were used to measure neutrophils' infiltration into the liver. The apoptotic index (TUNEL-positive cell number / total liver cell number × 100%) was calculated to assess hepatocelluar apoptosis. Finally, activation of NF-κB and phosphor-p38 expression in liver homogenates were analyzed with Western Blot (WB).ResultsOur findings showed that 1) CCK and CCK-AR were upregulated in our experimental model over time; 2) Treatment with CCK decreased sAST/sALT levels, inflammatory hepatic injury, neutrophil influx and hepatocelluar apoptosis, while proglumide aggravated hepatic injury.ConclusionThese findings support our hypothesis and suggest that CCK played a positive role in the ongoing inflammatory process leading to liver I/R injury.     

A short circulating peptide nanofiber as a carrier for tumoral delivery

The cellular interactions and in vivo distribution of the nanomaterials are known to be strongly influenced by their physiochemical properties. Here, we investigated and compared the biocompatibility, pharmacokinetics, and biodistribution of previously reported peptide-based nanofiber (NFP), with commercially available nanomaterials. The NFP was a 2-dimensional (2D) structure with an extremely narrow width (4 nm) and a controllable length (50 to 400 nm). NFP was found to be non-toxic, hemocompatible, and with a minimum uptake by macrophages. In vivo studies further demonstrated that NFP could be delivered to the tumor site more effectively, and within a very shorter period of time, than spherical nanoparticles. Importantly, the undelivered NFP was rapidly eliminated by renal clearance and, thus, avoiding its accumulation in the spleen or liver. Overall, our data suggested a new paradigm in drug delivery via using a short circulating NFP, rather than a long circulating 3D nanoparticle, as a delivery cargo.From the Clinical EditorIn this study, the role of small peptide-based nanocarriers is investigated for tumor-specific delivery, reporting excellent targeting properties and a favorable toxicity profile.     

Antitumor activity and toxicity relationship of annonaceous acetogenins

Annonaceous acetogenins (ACGs) are one of the most interesting classes of natural products appearing in the past two decades. Here, we studied the antitumor activity and toxicity relationship of ACGs including annosquamin B (1), bullatacin (2) and annosquatin B (3) in vivo. A single intraperitoneal (i.p.) injection of 100 μg/kg of annosquamin B, bullatacin and annosquatin B did not cause side effects in normal mice. Bullatacin treatment with five doses of 25 and 50 μg/kg in H₂₂ hepatoma cells bearing mice resulted in about 61% reduction in tumor growth with hematologic parameters increased significantly in normal mice. Annosquamin B and annosquatin B treatments with 10 doses of 25, 50 and 100 μg/kg in the H₂₂ hepatoma cells transplantation tumor model mice resulted in maximum 53.7% and 58.7% reduction in tumor growth, respectively, and did not cause severe side effects in normal mice. This study provided the evidence that adjacent bis-THF ACGs showed higher antitumor activity and toxicity than mono-THF and nonadjacent bis-THF ACGs in vivo. Furthermore, it was found that bullatacin led to liver and kidney toxicity via increasing calcium concentration, ROS production, and Bax expression and Bax/Bcl-2 ratio in rats with repeated treatment with bullatacin for 3 weeks.     

Effects of ischemia-reperfusion and pretreatment with mildronate on rat liver mitochondrial function

Mildronate (3-(2,2,2-trimethylhydrazinium) propionate), which is mostly used in cardiological practice and is considered an anti-ischemic drug, was designed to inhibit carnitine biosynthesis in order to prevent accumulation of cytotoxic intermediate products of fatty acid beta-oxidation. Recently it was shown that the mitochondrial respiratory chain may also be a target for mildronate action. In this study, we aimed to investigate whether mildronate can protect the liver against a 90-min normothermic ischemia/30-min reperfusion-induced mitochondrial dysfunction. Rats were pre-treated for one or two weeks with mildronate (100 mg/kg/day or 200 mg/kg/day) or Ringer solution and subjected to ischemia/reperfusion. We found that ischemia/reperfusion caused a decrease in mitochondrial State 3 respiration rate and in the respiratory control index (RCI), and an increase in State 2 respiration rate with succinate, glutamate + malate and palmitoyl-L-carnitine + malate. One or two weeks of pre-treatment of rats with different doses of mildronate did not reduce the ischemia/reperffusion-induced decrease in the State 3 respiration rate or RCI; however, a one week pre-treatment slightly diminished the increase in the State 2 respiration rate with glutamate + malate substrates. The leakage of the liver enzymes, aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase, was similar in both the untreated and pre-treated with mildronate groups. No steatotic livers were observed in any experimental groups after mildronate pre-treatment. In conclusion, 90 min of liver ischemia followed by a 30 min reperfusion has a deleterious effect on rat liver mitochondrial function. Mildronate pre-treatment of rats at doses of 100 or 200 mg/kg/day for one or two weeks did not prevent ischemia/reperfusion-induced mitochondrial dysfunction and liver injury.