In vivo bio-imaging using chlorotoxin-based conjugates

Surgical resection remains the primary component of cancer therapy. The precision required to successfully separate cancer tissue from normal tissue relies heavily on the surgeon's ability to delineate the tumor margins. Despite recent advances in surgical guidance and monitoring systems, intra-operative identification of these margins remains imprecise and directly influences patient prognosis. If the surgeon had improved tools to distinguish these margins, tumor progression and unacceptable morbidity could be avoided. In this article, we review the history of chlorotoxin and its tumor specificity and discuss the research currently being generated to target optical imaging agents to cancer tissue.     

Glutathione depletion by naphthalene in isolated hepatocytes and by naphthalene oxide in vivo

Previous studies have shown that naphthalene oxide and reactive naphthalene metabolites diffuse from intact, isolated hepatocytes. The amount of naphthalene oxide diffusing from the cells as a percentage of the total formed remained constant over a wide range of substrate concentrations, thus suggesting that depletion of glutathione might not be required prior to significant naphthalene oxide efflux. However, the relative intracellular versus extracellular covalent binding of reactive metabolites increased with increasing naphthalene concentrations, thereby suggesting that glutathione might be involved in modulating the extent of intracellular covalent binding. To examine this question in detail, intracellular glutathione levels were monitored in mouse hepatocytes incubated in the presence of various concentrations of naphthalene. Naphthalene produced a concentration- and time-dependent decrease in intracellular glutathione levels and, at higher concentrations, a marked decrease in the rate of glutathione efflux from hepatocytes. This decrease in hepatocellular glutathione levels correlated well with the shift in binding from predominantly extracellular to intracellular. Inclusion of glutathione and glutathione transferases in the cell incubation medium partially blocked the depletion of intracellular glutathione by naphthalene, thus suggesting that naphthalene oxide diffusing into the cell medium was partially responsible for intracellular glutathione depletion. Finally, in vivo administration of naphthalene oxide to mice produced a dose-dependent depletion of pulmonary but not hepatic or renal glutathione but only at doses that were greater than 75 mg/kg. These studies support the view that there is not a glutathione threshold for the efflux of naphthalene oxide from intact hepatocytes and suggest that naphthalene oxide is capable of diffusing into as well as out of isolated hepatocytes.     

In vitro formation of glutathione conjugates of the dimethylester of bilirubin.

Rat hepatic microsomes catalyzed the formation of two distinct glutathione conjugates of bilirubin dimethylester (DMB). The two conjugates were identical to those isolated from the bile of Gunn rats infused with DMB. The microsomal reaction was dependent on NADPH, oxygen and glutathione and was inhibited by nitrogen and the cytochrome P450 inhibitors metyrapone, 1-benzyl-imidazole, and alpha-naphthoflavone. Conjugate formation was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but not phenobarbital pretreatment. The rate of formation of conjugates was not affected by washings of the microsomal pellet or by the presence of superoxide dismutase and/or catalase. Cation fast atom bombardment mass spectrometry (FAB/MS) of the conjugates indicated a molecular ion of 937 atomic mass units (amu). Fragmentation revealed a loss of 307 amu, consistent with glutathione, and a residual mass of 629 amu suggesting a hydroxylated derivative of DMB (612 amu). Cation FAB/MS/MS of conjugates formed in vitro under an atmosphere of oxygen-16 and oxygen-18 demonstrated the incorporation of molecular oxygen by a difference of 2 amu in the respective molecular ions. Our results suggest that DMB is oxidized by the cytochrome P450 IA gene family to an epoxide intermediate which is then subsequently conjugated with glutathione.     

In vivo metabolism of aloemannan.

The metabolism of fluoresceinyl isothiocyanate labeled aloemannan (FITC-AM) was examined by p.o. and i.v. administration in mice at a dose of 120 mg/kg. Analysis of FITC-AM in urine and feces showed that FITC-AM (MW 500 KD) was metabolized into smaller molecules that mainly accumulated in the kidneys. AM was catabolized by the human intestinal microflora to catabolites 1 and 2 with molecular weights of 30 and 10 KD, respectively. Hydrolysis of AM showed hexosamine peaks on HPAE. The findings suggest that the immunomodulation of AM may come from not only neutral polysaccharides but also contaminated hexosamine in AM.     

In vivo murine studies on the biochemical mechanism of naphthalene cataractogenesis

The polycyclic aromatic hydrocarbon naphthalene is bioactivated by cytochromes P450 to an electrophilic epoxide intermediate, which subsequently is metabolized to naphthoquinones (NQ) and possibly to a free radical intermediate. These reactive intermediates may bind covalently to lenticular tissues, cause oxidant stress and/or lipid peroxidation, thereby initiating cataracts. To evaluate this hypothesis, male C57BL/6 or DBA/2 mice were treated with naphthalene or one of several naphthoquinone and naphthol metabolites, in the presence or absence of modulators of chemical bioactivation and detoxification. In C57BL/6 mice, cataracts were caused by naphthalene (500-2000 mg/kg ip) in a dose-dependent fashion. The incidence of naphthalene-induced cataracts was decreased by pretreatment with the P450 inhibitors SKF 525A and metyrapone, the antioxidants caffeic acid and vitamin E, the glutathione (GSH) precursor N-acetylcysteine, and the free radical spin trapping agent alpha-phenyl-N-t-butylnitrone (p less than 0.05). Naphthalene cataractogenicity was enhanced by pretreatment with the cytochrome P450 inducer phenobarbital and the GSH depletor diethyl maleate (DEM) (p less than 0.05), and was unaffected by pretreatment with the prostaglandin synthetase inhibitors aspirin or naproxen, or the epoxide hydrolase inhibitor trichloropropene oxide. Cataracts were initiated by 1,2-NQ and 1,4-NQ (5-250 mg/kg ip) in a dose-dependent fashion, with a molar potency about 10-fold higher than that for naphthalene. NQ cataractogenicity was enhanced by pretreatment with DEM (p less than 0.05). 1-Naphthol (56 to 562 mg/kg ip) demonstrated a cataractogenic potency intermediary to that for naphthalene and NQ. DBA/2 mice treated with naphthalene (2000 mg/kg ip), 1,4-NQ (65-250 mg/kg ip), 1,2-NQ (30-250 mg/kg ip), or DEM followed by 1,4-NQ (125 mg/kg ip) did not develop cataracts. These results suggest that naphthalene cataractogenesis in C57BL/6 mice requires P450-catalyzed bioactivation to a reactive intermediate, which may be the NQ and/or a free radical derivative, either of which is dependent upon GSH for detoxification.     

Drug protein conjugates—VI. Role of glutathione in the metabolism of captopril and captopril plasma protein conjugates

Previous metabolic studies of captopril suggest that the rapid dissociation of captopril-plasma protein conjugates in vivo is dependent upon endogenous thiols such as glutathione and cysteine. Consistent with this hypothesis, we have found that cysteine (0.06–3 mM) and glutathione (0.02–1 mM) cleave ¹⁴C-captopril-plasma protein conjugates in vitro. Dissociation of the drug-protein conjugate was accompanied by formation of the corresponcling mixed disulphide which indicates that the reaction proceeds via a spontaneous thiol-disulphide interchange. Administration of high doses (50–300 mg/kg) of CP produced a time-dependent and dose-dependent decrease in hepatic glutathione concentrations in the mouse and the rat. The depletion of glutathione observed was similar to that produced by equimolar doses of D-penicillamine and paracetamol. Acute and chronic (7 days) administration of captopril (100 mg/kg) produces the same (11–12%) depletion of hepatic glutathione. However, changes in liver function as determined by elevation of serum glutamic-pyruvic transaminase was only observed at doses of 200 and 300 mg/kg. Thus, although thiol-disuiphide interactions between captopril and plasma proteins may contribute to the perturbation of hepatic glutathione concentrations, it is unlikely that this process will be of toxicological significance during therapeutic administration of captopril.     

Transport of glutathione and glutathione conjugates by MRP1

Glutathione (GSH)-conjugated xenobiotics and GSH-conjugated metabolites (e.g. the cysteinyl leukotriene C4) must be exported from the cells in which they are formed before they can be eliminated from the body or act on their cellular targets. This efflux is often mediated by the multidrug resistance protein 1 (MRP1) transporter, which also confers drug resistance to tumour cells and can protect normal cells from toxic insults. In addition to drugs and GSH conjugates, MRP1 exports GSH and GSH disulfide, and might thus have a role in cellular responses to oxidative stress. The transport of several drugs and conjugated organic anions by MRP1 requires the presence of GSH, but it is not well understood how GSH (and its analogues) enhances transport. Site-directed mutagenesis studies and biophysical analyses have provided important insights into the structural determinants of MRP1 that influence GSH and GSH conjugate binding and transport.     

In vivo metabolism of retrorsine and retrorsine-N-oxide

The in vivo metabolism and excretion of the urinary metabolites from the pyrrolizidine alkaloids (PAs), retrorsine (RET) and retrorsine-N-oxide (RET-NO) have been studied in rats. Isatinecic acid (INA), pyrrolic metabolites, N-oxides and retronecine accounted for 31.0, 10.3, 10.8 and 0.39% of the administered RET. Predosing rats with triorthocresyl phosphate (TOCP), had no effect on the excretion of pyrrolic metabolites and INA. Phenobarbital (PB) increased the excretion of both pyrrolic metabolites and INA with a corresponding decrease in the excretion of RET and N-oxides; the retronecine levels remained unaltered. When RET-NO was administered i.p., the urinary levels of pyrrolic metabolites, INA and RET were decreased relative to those treated with RET. The p.o. administration of RET-NO produced significantly higher levels of pyrrolic metabolites, INA and RET. These results suggest that esterase hydrolysis plays a minor role in the formation of INA and that a common metabolic pathway may exist between pyrrolic metabolites and INA formation.     

In vivo nephrotoxic action of an isomeric mixture of S-(1-phenyl-2-hydroxyethyl)glutathione and S-(2-phenyl-2-hydroxyethyl)glutathione in Fischer-344 rats.

An isomeric mixture of S-[(1 and 2)-phenyl-2-hydroxyethyl]glutathione (PHEG), a glutathione conjugate of styrene, is moderately nephrotoxic. Its in vivo nephrotoxicity was characterized by significant elevations in the urinary excretion of glucose, gamma-glutamyl transpeptidase, glutamate dehydrogenase, N-acetyl-beta-D-glucosaminidase and lactic dehydrogenase 24 h after an i.v. administration of PHEG (0.5 mmol/kg) in male Fischer-344 rats. The histologic alterations consisted of moderate tubular damage with proximal tubule vacuolization and accumulation of tubular cast material, indicating an early sign of tubular necrosis. The data suggest that nephrotoxic injury induced by PHEG lies preferentially at the tubular region of the rat kidney involving several subcellular targets. The nephrotoxicity of PHEG was blocked by acivicin, a specific inhibitor of gamma-glutamyl transpeptidase, by phenylalanylglycine, an inhibitor of cysteinylglycine dipeptidase, as well as by probenecid, a competitive inhibitor of renal organic anion transport system. On the other hand, pretreatment with aminooxyacetic acid, a specific inhibitor of renal cysteine conjugate beta-lyase, failed to inhibit the nephrotoxicity of this glutathione conjugate. Similarly, prior administration of alpha-ketobutyrate, an inducer of renal cysteine conjugate beta-lyase, failed to potentiate its nephrotoxicity, suggesting an insignificant role of beta-lyase in such toxicity. A modest decline in renal cellular GSH due to PHEG but without any concomitant oxidation of GSH to GSSG and without any increase in lipid peroxidation indicates that oxidative stress may not be an important mechanism of its nephrotoxicity. Therefore, the following steps at least, are involved in the development of its nephrotoxicity: (1) renal tubular accumulation of PHEG via a probenecid-sensitive transport process; and (2) its renal metabolism via gamma-glutamyl transpeptidase and cysteinylglycine dipeptidase to the corresponding cysteine-S-conjugate.     

In vivo effects of fenthion on oxidative processes by the modulation of glutathione metabolism in the brain of Oreochromis niloticus

The present study was designed to understand the oxidative stress potential of fenthion, an organophosphate (OP) pesticide and its involvement in glutathione metabolism modulated buthionine sulfoximine (BSO, 50 mg/kg) and N-acetylcysteine (NAC, 100 mg/kg) in the brain of fish, Oreochromis niloticus. A sublethal fenthion concentration (0.45 mg/L) was applied for 24, 48, and 96 h together with injection with BSO or NAC; following treatment, recovery periods for 24, 48, and 96 h were allowed. Total glutathione (tGSH), oxidized glutathione (GSSG), lipid peroxidation, protein level, and GSH-related enzyme activities were analyzed by using spectrophotometric methods. Fenthion in applied concentration did not change GSH levels, but increased GSSG levels. BSO application in fenthion exposure caused a depletion in GSH, while increasing the GSSG levels. Glutathione peroxidase (GPx; EC 1.11.1.9) specific activity increased in fenthion-applied groups at 24-h treatment. gamma-Glutamylcysteinyl synthetase (gamma-GCS; EC 6.3.2.2) was not detected in the brain. NAC injection in fenthion treatment decreased GSH and increased GSSG levels and GST activity. In conclusion, fenthion in sublethal concentration induced an oxidative stress processes in brain. BSO application provided an evidence for the involvement of fenthion in GSH metabolism. NAC elevated the fenthion-induced effects in spite of its antioxidant properties. Recovery period for 96 h was not adequate to eliminate the fenthion-induced changes.     

In vitro metabolism of naphthalene by human liver microsomal cytochrome P450 enzymes.

The polycyclic aromatic hydrocarbon naphthalene is an environmental pollutant, a component of jet fuel, and, since 2000, has been reclassified as a potential human carcinogen. Few studies of the in vitro human metabolism of naphthalene are available, and these focus primarily on lung metabolism. The current studies were performed to characterize naphthalene metabolism by human cytochromes P450. Naphthalene metabolites from pooled human liver microsomes (pHLMs) were trans-1,2-dihydro-1,2-naphthalenediol (dihydrodiol), 1-naphthol, and 2-naphthol. Metabolite production generated Km values of 23, 40, and 116 microM And Vmax values of 2860, 268, and 22 pmol/mg protein/min, respectively. P450 isoform screening of naphthalene metabolism identified CYP1A2 as the most efficient isoform for producing dihydrodiol and 1-naphthol, and CYP3A4 as the most effective for 2-naphthol production. Metabolism of the primary metabolites of naphthalene was also studied to identify secondary metabolites. Whereas 2-naphthol was readily metabolized by pHLMs to produce 2,6- and 1,7-dihydroxynaphthalene, dihydrodiol and 1-naphthol were inefficient substrates for pHLMs. A series of human p450 isoforms was used to further explore the metabolism of dihydrodiol and 1-naphthol. 1,4-Naphthoquinone and four minor unknown metabolites from 1-naphthol were observed, and CYP1A2 and 2D6*1 were identified as the most active isoforms for the production of 1,4-naphthoquinone. Dihydrodiol was metabolized by P450 isoforms to three minor unidentified metabolites with CYP3A4 and CYP2A6 having the greatest activity toward this substrate. The metabolism of dihydrodiol by P450 isoforms was lower than that of 1-naphthol. These studies identify primary and secondary metabolites of naphthalene produced by pHLMs and P450 isoforms.     

In vivo real-time confocal microscopy for target-specific delivery of hyaluronic acid-quantum dot conjugates

Hyaluronic acid (HA), which is a biocompatible, biodegradable, and linear polysaccharide in the body, has been widely used for various biomedical applications. In this work, real-time bioimaging for target-specific delivery of HA derivatives was carried out using quantum dots (QDs). In vitro confocal microscopy of HA-QD conjugates confirmed the intracellular delivery of HA derivatives to B16F1 cells with HA receptors by HA-receptor-mediated endocytosis. Furthermore in vivo real-time confocal microscopy of HA-QD conjugates successfully visualized the target specific delivery and accumulation of HA-QD conjugates from the fluorescence-labeled blood vessels to the liver tissues. The authors could confirm the feasibility of HA derivatives as a target-specific intracellular drug-delivery carrier for the treatment of liver diseases and the in vivo real-time confocal microscopy as a new bioimaging tool for various drug-delivery applications.From the Clinical EditorThis study demonstrates the possibility of labeling hyaluronic acid with quantum dots for visualization and for targeted intracellular drug delivery in liver disease models.     

In vivo metabolism of the cardiovascular toxin, allylamine

Previous evidence from this laboratory demonstrated that allylamine, a known cardiovascular toxin, is metabolized in vitro to acrolein, which has been hypothesized to act as a distal toxin. In this study, 3-hydroxypropylmercapturic acid was isolated and identified by MS, NMR, and 2D-NMR spectroscopy as the sole urinary metabolite of allylamine metabolism in vivo. Parallel experiments showed reduced glutathione (GSH) depletion in several organs (most marked in aorta, blood, and lung), which is consistent with GSH conjugation of the proposed acrolein intermediate. These findings indicate that allylamine was metabolized in vivo to a highly reactive aldehyde which was converted to a mercapturic acid through a GSH conjugation pathway; the exact mechanisms of cellular damage remain unclear.