Effects of menthofuran, a monoterpene furan on rat liver microsomal enzymes, in vivo

Oral administration (250 mg/kg) of menthofuran, a monoterpene furan, to rats once daily for 3 days caused hepatotoxicity as judged by a significant increase in serum glutamate pyruvate transaminase (SGPT) and decreases in glucose-6-phosphatase and aminopyrine N-demethylase activities. Administration of menthofuran also resulted in a decrease in the levels of liver microsomal cytochrome P-450, whereas cytochrome b(5) and NAD(P)H-cytochrome c reductase activities were not affected. These effects of menthofuran were both dose- and time-dependent. Pretreatment of rats with phenobarbital (PB) prior to menthofuran treatment potentiated hepatotoxicity suggesting that a PB-induced cytochrome P-450 catalyzed the formation of reactive metabolite(s) responsible for the hepatotoxicity.

In vitro and in vivo effects of methyl isocyanate on rat brain mitochondrial respiration

The present study deals with the in vitro and in vivo effects of methyl isocyanate (MIC) on rat brain mitochondrial function. Addition of MIC to tightly coupled brain mitochondria in vitro resulted in a mild stimulation of state 4 respiration, abolition of respiratory control, decrease in ADP/0 ratio, and inhibition of state 3 oxidation. The oxidation of NAD+-linked substrates (glutamate + malate) was more sensitive (fourfold) to the inhibitory action of MIC than succinate while cytochrome oxidase was unaffected. Administration of MIC subcutaneously at a lethal dose affected respiration only with glutamate + malate as the substrate (site I) and caused a 20% decrease in state 3 oxidation leading to a significant decrease in respiratory control index while state 4 respiration and ADP/O ratio remained unaffected. As both the malondialdehyde and iron contents of brain mitochondria were not altered, it may be inferred that the observed in vivo inhibition of state 3 oxidation is induced by MIC through systemic stagnant hypoxia leading to ischemia of brain, which further contributes to the cerebral hypoxia.

In vivo and in vitro studies on the differential role of luteinizing hormone and follicle-stimulating hormone in regulating follicular function in the bonnet monkey (Macaca radiata) using specific gonadotropin antibodies

Although a distinct need for FSH in the regulation of follicular maturation in the primate is well recognized, it is not clear how FSH controls the functionality of different cellular compartments of the follicle. It is also not evident whether there is a requirement for LH in follicular maturation in the primate. In the first part of the present study, female bonnet monkeys were administered a well-characterized ovine (o) LH antiserum to neutralize endogenous monkey LH for different periods during the follicular phase, and the effect on the overall follicular maturation process was assessed by analyzing serum estrogen (E) and progesterone (P) profiles. Neither continuous LH deprivation from Day 8 of the cycle nor deprivation of LH on any one day between Days 6 and 10 had a significant effect on serum E and P profiles and the follicular maturation process. The period for which the antiserum was effective was dependent upon the dose injected; 1 ml of the antiserum given on Day 8 blocked ovulation but not follicular maturation. To assess the effect of deprivation of LH/FSH at the cellular level, animals were deprived in vivo of LH (on Days 8 and 9 of the cycle) or of FSH (on Day 6 of the cycle) by injection of highly characterized hCG and ovine (o) FSH antisera, respectively; the in vitro responsiveness of granulosa and thecal cells isolated on Day 10 from these animals was then determined.(ABSTRACT TRUNCATED AT 250 WORDS)

Imaging in vivo Neuronal Transport in Genetic Model Organisms Using Microfluidic Devices

Microfluidic devices have been developed for imaging behavior and various cellular processes in Caenorhabditis elegans, but not subcellular processes requiring high spatial resolution. In neurons, essential processes such as axonal, dendritic, intraflagellar and other long-distance transport can be studied by acquiring fast time-lapse images of green fluorescent protein (GFP)-tagged moving cargo. We have achieved two important goals in such in vivo studies namely, imaging several transport processes in unanesthetized intact animals and imaging very early developmental stages. We describe a microfluidic device for immobilizing C. elegans and Drosophila larvae that allows imaging without anesthetics or dissection. We observed that for certain neuronal cargoes in C. elegans, anesthetics have significant and sometimes unexpected effects on the flux. Further, imaging the transport of certain cargo in early developmental stages was possible only in the microfluidic device. Using our device we observed an increase in anterograde synaptic vesicle transport during development corresponding with synaptic growth. We also imaged Q neuroblast divisions and mitochondrial transport during early developmental stages of C. elegans and Drosophila, respectively. Our simple microfluidic device offers a useful means to image high-resolution subcellular processes in C. elegans and Drosophila and can be readily adapted to other transparent or translucent organisms.

A method for in vivo [32P]phosphate labelling of testis proteins

The direct intratesticular injection of [P-32]phosphate resulted in 4-9 times more labelling of rat testis proteins compared to the conventional method of in vitro incubation. Moreover this is a simple technique requiring minimum (7-10 times less) radioactive phosphate and is less hazardous.

In vitro and in vivo effect of methyl isocyanate on rat liver mitochondrial respiration

Previous work has shown that irrespective of the route of exposure methyl isocyanate (MIC) caused acute lactic acidosis in rats (Jeevaratnam et al., Arch. Environ. Contam. Toxicol. 19, 314�319, 1990) and the hypoxia was of stagnant type due to tissue hypoperfusion resulting from hypovolemic hypotension in rabbits administered MIC subcutaneously (Jeevarathinam et al., Toxicology 51, 223�240, 1988). The present study was designed to investigate whether MIC could induce histotoxic hypoxia through its effects on mitochondrial respiration. Male Wistar rats were used for liver mitochondrial and submitochondrial particle (SMP) preparation. Addition of MIC to tightly coupled mitochondria in vitro resulted in stimulation of state 4 respiration, abolition of respiratory control, decrease in ADP/O ratio, and inhibition of state 3 oxidation. The oxidation of NAD+-linked substrates (glutamate + malate) was more sensitive (fiveto sixfold) to the inhibitory action of MIC than succinate while cytochrome oxidase remained unaffected. MIC induced twofold delay in the onset of anerobiosis, and cytochrome b reduction in SMP with NADH in vitro confirms inhibition of electron transport at complex I region. MIC also stimulated the ATPase activity in tightly coupled mitochondria while lipid peroxidation remained unaffected. As its hydrolysis products, methylamine and N,N?-dimethylurea failed to elicit any change in vitro; these effects reveal that MIC per se acts as an inhibitor of electron transport and a weak uncoupler. Administration of MIC sc at lethal dose caused a similar change only with NAD+-linked substrates, reflecting impairment of mitochondrial respiration at complex I region and thereby induction of histotoxic hypoxia in vivo.

Copper(I) complexes of modified nucleobases and vitamin B3 as potential chemotherapeutic agents: In vitro and in vivo studies

Three new complexes of Cu(I) have been synthesized using ancillary ligands like thiopyrimidine (tp) a modified nucleobase, and nicotinamide (nie) or vitamin B3, and characterized by spectroscopy and X-ray crystallography. In vitro cytotoxicity studies of the complexes on various human cancer cell lines such as Colo295, H226, HOP62, K562, MCF7 and T24 show that Cu(PPh3)(2)(tp)Cl] and Cu(PPh3)(2)(tp)ClO4 (2) have in vitro cytotoxicity comparable to cisplatin. Complex Cu(nic)(3)PPh3]ClO4 (3) is non-toxic and increases the life span by about 55 % in spontaneous breast tumor model. DNA binding and cleavage studies show that complex (3) binds to calf thymus DNA with an apparent binding constant of 5.9 x 10(5)M and completely cleaves super-coiled DNA at a concentration of 400 mu M, whereas complexes (1) and (2) do not bind DNA and do not show any cleavage even at 1200 mu M. Thus, complex (3) may exhibit cytotoxicity Via DNA cleavage whereas the mechanism of cytotoxicity of (1) and (2) probably involves a different pathway.

In vitro and in vivo regulation of assimilatory nitrite reductase from Candida utilis

The nitrate assimilation pathway in Candida utilis, as in other assimilatory organisms, is mediated by two enzymes: nitrate reductase and nitrite reductase. Purified nitrite reductase has been shown to be a heterodimer consisting of 58- and 66-kDa subunits. In the present study, nitrite reductase was found to be capable of utilising both NADH and NADPH as electron donors. FAD, which is an essential coenzyme, stabilised the enzyme during the purification process. The enzyme was modified by cysteine modifiers, and the inactivation could be reversed by thiol reagents. One cysteine was demonstrated to be essential for the enzymatic activity. In vitro, the enzyme was inactivated by ammonium salts, the end product of the path way, proving that the enzyme is assimilatory in function. In vivo, the enzyme was induced by nitrate and repressed by ammonium ions. During induction and repression, the levels of nitrite reductase mRNA, protein, and enzyme activity were modulated together, which indicated that the primary level of regulation of this enzyme was at the transcriptional level. When the enzyme was incubated with ammonium salts in vitro or when the enzyme was assayed in cells grown with the same salts as the source of nitrogen, the residual enzymatic activities were similar. Thus, a study of the in vitro inactivation can give a clue to understanding the mechanism of in vivo regulation of nitrite reductase in Candida utilis.

In vivo clearance of Japanese encephalitis virus by adoptively transferred virus specific cytotoxic T lymphocytes

Japanese encephalitis virus (JEV) is a positive stranded RNA virus that belongs to the flavivirus group, JEV infection damages the central nervous system (CNS) and is one of the main causative agents of acute encephalitis, H-2 restricted virus-specific cytotoxic T lymphocytes (CTL) have been generated specifically against JEV in our laboratory and these CTL have been shown to protect mice against lethal challenge with JEV, Virus replication was found to be inhibited in the brains of animals that mere adoptively transferred with JEV specific CTL as revealed by immunohistological staining as,veil as viral plaque assays. We further show that virus specific CTL could be recovered from such protected mice as long as 45 days after adoptive transfer.

Non-viral ex vivo hepatic gene transfer by in situ lipofection of liver and intraperitoneal transplantation of hepatocytes

Perfusion of liver with plasmid DNA-lipofectin complexes via the portal vein results in efficient accumulation of the vector in hepatocytes. Such hepatocytes, when administered intraperitoneally into a hepatectomized rat, repopulate the liver and express the transgene efficiently. This procedure obviates the need for large-scale hepatocyte culture for ex vivo gene transfer. Further, intraperitoneal transplantation is a simple and cost-effective strategy of introducing genetically modified hepatocytes into liver. Thus, in situ lipofection of liver and intraperitoneal transfer of hepatocytes can be developed into a novel method of non-viral ex vivo gene transfer technique that has applications in the treatment of metabolic disorders of liver and hepatic gene therapy.

In Vitro and in Vivo Anticancer Activity of Copper Bis(thiosemicarbazone) Complexes

Neutral and cationic copper bis(thiosemicarbazone) complexes bearing methyl, phenyl, and hydrogen, on the diketo-backbone of the ligand have been synthesized. All of them were characterized by spectroscopic methods and in three cases by X-ray crystallography. In vitro cytotoxicity studies revealed that they are cytotoxic unlike the corresponding zinc complexes. Copper complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) (GTSCH(2)) are the most cytotoxic complexes against various human cancer cell lines, with a potency similar to that of the anticancer drug adriamycin and up to 1000 fold higher than that of the corresponding zinc complex. Tritiated thymidine incorporation assay revealed that Cu(GTSC) and Cu(GTSCHCl) inhibit DNA synthesis substantially. Cell cycle analyses showed that Cu(GTSC) and Cu(GTSCHCl) induce apoptosis in HCT116 cells. The Cu(GTSCHCl) complex caused distinct DNA cleavage and Topo II alpha inhibition unlike that for Cu(GTSC). In vivo administration of Cu(GTSC) significantly inhibits tumor growth in HCT116 xenografts in nude mice.

Metabolites of PPI-2458, a selective, irreversible inhibitor of methionine aminopeptidase-2: structure determination and In vivo activity

The natural product fumagillin exhibits potent antiproliferative and antiangiogenic properties. The semisynthetic analog PPI-2458, (3R,4S,5S,6R)-5-methoxy-4-(2R,3R)-2-methyl-3-(3-methylbut-2-enyl) oxiran-2-yl]-1-oxaspiro2.5]octan-6-yl] N-(2R)-1-amino-3-methyl-1-oxobutan-2-yl]carbamate, demonstrates rapid inactivation of its molecular target, methionine aminopeptidase-2 (MetAP2), and good efficacy in several rodent models of cancer and inflammation with oral dosing despite low apparent oral bioavailability. To probe the basis of its in vivo efficacy, the metabolism of PPI-2458 was studied in detail. Reaction phenotyping identified CYP3A4/5 as the major source of metabolism in humans. Six metabolites were isolated from liver microsomes and characterized by mass spectrometry and nuclear resonance spectroscopy, and their structures were confirmed by chemical synthesis. The synthetic metabolites showed correlated inhibition of MetAP2 enzymatic activity and vascular endothelial cell growth. In an ex vivo experiment, MetAP2 inhibition in white blood cells, thymus, and lymph nodes in rats after single dosing with PPI-2458 and the isolated metabolites was found to correlate with the in vitro activity of the individual species. In a phase 1 clinical study, PPI-2458 was administered to patients with non-Hodgkin lymphoma. At 15 mg administered orally every other day, MetAP2 in whole blood was 80% inactivated for up to 48 hours, although the exposure of the parent compound was only similar to 10% that of the summed cytochrome P450 metabolites. Taken together, the data confirm the participation of active metabolites in the in vivo efficacy of PPI-2458. The structures define a metabolic pathway for PPI-2458 that is distinct from that of TNP-470 ((3R, 4S, 5S, 6R)-5-methoxy-4-(2R, 3R)-2-methyl-3-(3-methylbut-2-enyl)oxiran-2-yl]-1-oxaspiro2.5]octan-6 -yl] N-(2-chloroacetyl)carbamate). The high level of MetAP2 inhibition achieved in vivo supports the value of fumagillin-derived therapeutics for angiogenic diseases.

In vitro cytotoxicity and in vivo osseointergration properties of compression-molded HDPE-HA-Al2O3 hybrid biocomposites

The aim of this study was to investigate the in vivo biocompatibility in terms of healing of long segmental bone defect in rabbit model as well as in vitro cytotoxicity of eluates of compression-molded High density polyethylene (HDPE)hydroxyapatite (HA)-aluminum oxide (Al2O3) composite-based implant material. Based on the physical property in terms of modulus and strength properties, as reported in our recent publication, HDPE-40 wt % HA and HDPE-20 wt % HA-20 wt % Al2O3 hybrid composites were used for biocompatibility assessment. Osteoblasts cells were cultured in conditioned media, which contains varying amount of composite eluate (0.01, 0.1, and 1.0 wt %). In vitro, the eluates did not exhibit any significant negative impact on proliferation, mineralization or on morphology of human osteoblast cells. In vivo, the histological assessment revealed neobone formation at the bone/implant interface, characterized by the presence of osteoid and osteoblasts. The observation of osteoclastic activity indicates the process of bone remodeling. No inflammation to any noticeable extent was observed at the implantation site. Overall, the combination of in vitro and in vivo results are suggestive of potential biomedical application of compression-molded HDPE- 20 wt % HA- 20 wt % Al2O3 composites to heal long segmental bone defects without causing any toxicity of bone cells.

A novel adeno-associated virus serotype (AAV)-8 capsid mutant improves human coagulation factor IX expression in vivo

Recombinant AAV-8 vectors have shown significant promise for hepatic gene therapy of hemophilia B. However, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses. We hypothesized that AAV8 during its intracellular trafficking, are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery and modification of specific serine/threonine kinase or ubiquitination targets on AAV8 capsid (Fig.1A) may improve its transduction efficiency. To test this, point mutations at specific serine (S)/threonine (T) > alanine (A) or lysine (K)>arginine (R) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant(S276A, S501A, S671A, T251A and K137R) capsids were evaluated for their liver transduction efficiency at a dose of 5 X 1010 vgs/ animal in C57BL/6 mice in vivo. The best performing mutant was found to be the K137R vector in terms of either the gene expression (46-fold) or the vector copy numbers in the hepatocytes (22-fold) compared to WT-AAV8 (Fig.1B). The K137R-AAV8 vector that showed significantly decreased ubiquitination of the viral capsid had reduced activation of markers of innate immune response [IL-6, IL-12, tumor necrosis factor α, Kupffer cells and TLR-9]. In addition, animals injected with the K137R mutant also demonstrated decreased (2-fold) levels of cross-neutralizing antibodies when compared to animals that received the WT-AAV8 vector. To study further the utility of the novel AAV8-K137R mutant in a therapeutic setting, we delivered human coagulation factor IX (h.FIX) under the control of liver specific promoters (LP1 or hAAT) at two different doses (2.5x10^10 and 1x10^11 vgs per mouse) in 8-12 weeks old male C57BL/6 mice. As can be seen in Fig.1C/D, the circulating levels of h.FIX were higher in all the K137R-AAV8 treated groups as compared to the WT-AAV8 treated groups either at 2 weeks (62% vs 37% for hAAT constructs and 47% vs 21% for LP1 constructs) or 4 weeks (78% vs 56% for hAAT constructs and 64% vs 30% for LP1 constructs) post hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel vector for potential gene therapy of hemophilia B.