Influence of uranium(VI) speciation for the evaluation of in vitro uranium cytotoxicity on LLC-PK1 cells

Very few data are available concerning the in vitro toxicity of uranium. In this work, we have determined the experimental chemical conditions permitting the observation of uranium(VI) cytotoxicity on LLC-PK1 cells. Uranium solutions made either by dissolving uranyl acetate or nitrate crystals, or by complexing uranium with bicarbonate, phosphate or citrate ligands, were prepared and tested. Experiments demonstrated that only uranium solutions containing citrate and bicarbonate ligands concentrations tenfold higher than the metal, were soluble in the cell culture medium. Cytotoxicity studies of all these uranium compounds were performed on LLC-PK1 cells and compared using LDH release, neutral red uptake and MTT assays. Dose dependent cytotoxicity curves were only obtained with uranium-bicarbonate medium. This study has revealed a toxicity of uranium-bicarbonate complexes for 24 h expositions and for concentrations ranging from 7 x 10(-4)-10(-3) M, under these conditions, the CI50 (cytotoxicity index) was evaluated between 8.5 and 9 x 10(-4) M. In contrast, we noticed a lack of cytotoxicity response for uranium(VI)-citrate complexes. Electron transmission microscopy studies revealed, when LLC-PK1 cells were exposed to the uranium-bicarbonate system, that uranium penetrated and precipitated within the cytoplasmic compartment. Morphological studies conducted with citrate complexes did not show any cellular intake of uranium.     

Role of the sodium-dependent phosphate co-transporters and of the phosphate complexes of uranyl in the cytotoxicity of uranium in LLC-PK1 cells

Although uranium is a well-characterized nephrotoxic agent, very little is known at the cellular and molecular level about the mechanisms underlying the uptake and toxicity of this element in proximal tubule cells. The aim of this study was thus to characterize the species of uranium that are responsible for its cytotoxicity and define the mechanism which is involved in the uptake of the cytotoxic fraction of uranium using two cell lines derived from kidney proximal (LLC-PK(1)) and distal (MDCK) tubule as in vitro models. Treatment of LLC-PK(1) cells with colchicine, cytochalasin D, concanavalin A and PMA increased the sodium-dependent phosphate co-transport and the cytotoxicity of uranium. On the contrary, replacement of the extra-cellular sodium with N-methyl-D-glucamine highly reduced the transport of phosphate and the cytotoxic effect of uranium. Uranium cytotoxicity was also dependent upon the extra-cellular concentration of phosphate and decreased in a concentration-dependent manner by 0.1-10 mM phosphonoformic acid, a competitive inhibitor of phosphate uptake. Consistent with these observations, over-expression of the rat proximal tubule sodium-dependent phosphate co-transporter NaPi-IIa in stably transfected MDCK cells significantly increased the cytotoxicity of uranium, and computer modeling of uranium speciation showed that uranium cytotoxicity was directly dependent on the presence of the phosphate complexes of uranyl UO(2)(PO(4))(-) and UO(2)(HPO(4))(aq). Taken together, these data suggest that the cytotoxic fraction of uranium is a phosphate complex of uranyl whose uptake is mediated by a sodium-dependent phosphate co-transporter system.     

Heat shock proteins and acquired resistance to uranium nephrotoxicity

Previous studies have demonstrated that prior exposures to uranium can produce acquired resistance to uranium nephrotoxicity. In this study, the potential role for heat shock proteins (Hsps) in acquired resistance to uranium nephrotoxicity was explored. Pretreatment of male Sprague-Dawley rats with a conditioning dose of uranyl acetate (5 mg/kg, i.p.) was found to diminish the severity of proximal convoluted tubule necrosis and azotemia produced by a subsequent, higher uranyl acetate dose (10 mg/kg, i.p., 10 days after the conditioning dose). Kidney homogenates from rats euthanized at the end of the conditioning period were found to contain elevated levels of Hsp25, Hsp32, and Hsp70i, but not Hsc70. Immunochemical staining of renal sections for Hsp25 and Hsp70i revealed that these proteins were prominently expressed in tubular epithelial cells in uranyl acetate pretreated animals. Morphological characteristics and staining for proliferating cell nuclear antigen (PCNA) indicated that the cells expressing high levels of Hsps were regenerating. In RK3E and LLC-PK1 renal epithelial cells in culture, Hsp induction by thermal pretreatment did not afford protection from uranyl acetate cytotoxicity. Further, treatment of RK3E and LLC-PK1 cells with uranyl acetate did not result in induction of Hsps, as occurs with other nephrotoxic heavy metals. These observations suggest that while stress proteins are elevated in acquired resistance to uranyl acetate in vivo, they are not responsible for diminished uranium nephrotoxicity but are an epiphenomenon of tubular epithelial regeneration.     

Influence of uranium speciation on normal rat kidney (NRK-52E) proximal cell cytotoxicity

Uranium is a naturally occurring heavy metal. Its extensive use in the nuclear cycle and for military applications has focused attention on its potential health effects. Acute exposures to uranium are toxic to the kidneys where they mainly cause damage to proximal tubular epithelium. The purpose of this study was to investigate the biological consequences of acute in vitro uranyl exposure and the influence of uranyl speciation on its cytotoxicity. NRK-52E cells, representative of rat kidney proximal epithelium, were exposed to uranyl-carbonate and -citrate complexes, which are the major complexes transiting through renal tubules after acute in vivo contamination. Before NRK-52E cell exposure, these complexes were diluted in classical or modified cell culture media, which can possibly modify uranyl speciation. In these conditions, uranium cytotoxicity appears after 16 h of exposure. The CI50 cytotoxicity index, the uranium concentration leading to 50% dead cells after 24 h of exposure, is 500 microM (+/-100 microM) and strongly depends on uranyl counterion and cell culture medium composition. Computer modeling of uranyl speciation is reported, enabling one to draw a parallel between uranyl speciation and its cytotoxicity.     

Influence of cadmium speciation for the evaluation of in vitro cadmium toxicity on LLC-PK(1) cells.

The main objective of the present work was to assess the potentiality of in vitro models to improve our understanding of cadmium-induced toxicity, especially on epithelial renal cells. Indeed cadmium, a potent toxic metal, poses a serious environmental threat and the mechanisms of its renal toxicity need to be clarified. Cytotoxicity studies presented here were performed in a tubular proximal original established porcine kidney cell line (LLC-PK(1)). We have compared cytotoxicity induced by different chemical cadmium forms in LLC-PK(1) cells as a function of media cell culture pH and protein content. Cadmium stock solutions were prepared either by dissolving cadmium chloride or cadmium sulphate with increasing protein concentrations in the media cell culture. Its pH was monitored during experiments. Cytotoxicity was measured by neutral red uptake after 24 h of exposure. Dose-dependent cytotoxicity curves, calculated with REGTOX, were systematically correlated with pH and protein content. Experiments in vitro revealed that cadmium was dose-dependently toxic for LLC-PK(1) for concentrations ranging from 10(-4) to 10(-6) M. We have noticed a lack of influence of the media cell culture pH on the cadmium cytotoxicity. REGTOX determines closely the EC(50) values but EC(50)CdCl(2)>EC(50)CdSO(4) and cadmium have been assayed with an inductively coupled atomic emission spectrometer (ICP/AES) directly in the media cell culture and the cellular pellet.     

Distribution of soluble uranium in the nuclear cell compartment at subtoxic concentrations

Uranium is naturally found in the environment, and its extensive use results in an increased risk of human exposure. Kidney cells have mainly been used as in vitro models to study effects of uranium exposure, and very little about the effects on other cell types is known. The aim of this study was to assess the impact of depleted uranium exposure at the cellular level in human kidney (HEK-293), liver (HepG2), and neuronal (IMR-32) cell lines. Cytotoxicity studies showed that these cell lines reacted in a roughly similar manner to depleted uranium exposure, responding at a cytotoxicity threshold of 300-500 μM. Uranium was localized in cells with secondary ion mass spectrometry technology. Results showed that uranium precipitates at subtoxic concentrations (>100 μM). With this approach, we were able for the first time to observe the soluble form of uranium in the cell at low concentrations (10-100 μM). Moreover, this technique allows us to localize it mainly in the nucleus. These innovative results raise the question of how uranium penetrates into cells and open new perspectives for studying the mechanisms of uranium chemical toxicity.     

Role of the sodium-dependent phosphate cotransporters and absorptive endocytosis in the uptake of low concentrations of uranium and its toxicity at higher concentrations in LLC-PK1 cells.

It has been suggested that uranium uptake and toxicity could be mediated by endocytosis and/or the type IIa sodium-dependent phosphate cotransporter (NaPi-IIa). The aim of this study was therefore to characterize in vitro the role of these two cellular mechanisms in the uptake and toxicity of low (200-3200 nM) and high (0.5 and 0.8 mM) concentrations of uranium, respectively. At low concentrations, uranium uptake in LLC-PK(1) cells was saturable (V(max) = 3.09 +/- 0.22 ng/mg protein) and characterized by a K(0.5) of 1022 +/- 63 nM and a Hill coefficient of 3.0 +/- 0.4. The potential involvement of endocytosis and NaPi-IIa in the uptake of uranium was assessed by the use of various drugs and culture conditions known to alter their relative activity, and (233)uranium uptake was monitored. Interestingly, the inhibitory effect of colchicine, cytochalasin D, phorbol 12-myristate 13-acetate, and chlorpromazine on endocytosis was highly correlated with their effect on uranium uptake, a relationship that was not true when the NaPi-IIa transport system was studied. Whereas the competitive inhibition of the NaPi-IIa by phosphonoformic acid (PFA) significantly decreased uranium uptake, this effect was not reproduced when NaPi-IIa inhibition was mediated by the replacement of extracellular Na(+) with N-methyl-D-glucamine. Uranium uptake was also not significantly altered when NaPi-IIa expression was stimulated in MDCK cells. More surprisingly, we observed by transmission electron microscopy that uranium cytotoxicity was dependent upon the extent of its intracellular precipitation, but not on its intracellular content, and was suppressed by PFA. In conclusion, our results suggest that low-dose uranium uptake is mainly mediated by absorptive endocytosis, and we propose PFA as a potential uranium chelator.     

Bioactivation mechanism of S-(3-oxopropyl)-N-acetyl-L-cysteine, the mercapturic acid of acrolein.

S-(3-Oxopropyl)glutathione, the glutathione conjugate of acrolein, has been reported to be nephrotoxic. The objective of the present studies was to investigate the bioactivation mechanism of the analogues S-(3-oxopropyl)-N-acetyl-L-cysteine (1) and S-(3-oxopropyl)-N-acetyl-L-cysteine S-oxide (2) and to test the hypothesis that the cytotoxicity of 1 is associated with its latent potential to release acrolein in kidney cells. Mechanistic considerations indicated that sulfoxidation of sulfide 1 to form S-oxide 2 and a subsequent general-base-catalyzed beta-elimination reaction would release the cytotoxin acrolein. Hence the release of acrolein from 1 and 2 was studied in chemical systems, and their cytotoxicity was investigated in cultured LLC-PK1 cells and in isolated rat renal proximal tubular cells. Acrolein formation from S-oxide 2, but not from sulfide 1, was observed under basic conditions and with phosphate as the base. Kinetic analysis indicated that a general-base-catalyzed reaction was involved. Both S-conjugates 1 and 2 were cytotoxic in LLC-PK1 cells and in isolated rat renal proximal tubular cells, and the cytotoxicity of sulfide 1, but not of S-oxide 2, in isolated renal proximal tubular cells was reduced in presence of methimazole, an inhibitor of the flavin-containing monooxygenase. These findings indicate that the cytotoxicity of S-conjugate 1 is associated with a novel bioactivation mechanism that involves sulfoxidation followed by a general-base-catalyzed elimination of acrolein from S-oxide 2.     

Phosphate regulates uranium(VI) toxicity to Lemna gibba L. G3

The influence of phosphate on the toxicity of uranium to Lemna gibba G3 was tested in semicontinuous culture with synthetic mine water developed as an analogue of surface water of two abandoned uranium mining and ore processing sites in Saxony, Germany. Six concentrations of uranium were investigated under five different supply regimes of PO(4) (3-) at constant pH (7.0 +/- 0.5) and alkalinity (7.0 +/- 1.6 mg L(-1) total CO(3) (2-)). The results showed significant inhibition of specific growth rates in cultures exposed to the highest uranium concentrations (3500 and 7000 microg U L(-1)) at lowest PO(4) (3-) supply of 0.01 mg L(-1). An increase of phosphate concentration from 0.01 to 8.0 mg L(-1) resulted in an increase of EC(50) from 0.9 +/- 0.2 to 7.4 +/- 1.9 mg L(-1) (significant with Student's t test, P > 0.05). The accumulation of uranium in L. gibba increased exponentially with the increase in uranium concentration in cultures with 0.01 and 0.14 mg PO(4) (3-) L(-1). Accumulation also increased significantly when PO(4) (3-) supply was increased from 0.14 to 1.36 mg PO(4) (3-) L(-1) for all uranium concentrations. However, as the supply of PO(4) (3-) gradually increased from 1.36 to 8.0 mg PO(4) (3-) L(-1), uranium bioaccumulation increased slightly but insignificantly before leveling off. Uranium speciation modeling with PhreeqC geochemical code predicted increases in the proportions of uranyl phosphate species when PO(4) (3-) concentrations increase in the media. Most of these uranyl phosphate species have a high probability of precipitation [saturation indices (SI) > 0.93]. Therefore, the alleviation of uranium toxicity to L. gibba with phosphates is due to interactions among components of the media, mainly uranyl and phosphate which results in precipitation. Consequently, bioavailable fractions of uranium to L. gibba are reduced. This might explain lack of consistent EC(50) values for uranium to most aquatic organisms.     

Effect of antioxidants on para-aminophenol-induced toxicity in LLC-PK1 cells

The present studies were designed to investigate the susceptibility of LLC-PK1 cells to cytotoxicity induced by para-aminophenol (PAP) and the ability of antioxidants to prevent PAP-induced cytotoxicity. LLC-PK1 cells were incubated for 4 h with varying concentrations of PAP (0-0.2 mM). Incubation was continued for 20 h and viability was monitored at 24 h after initial exposure to PAP. For coincubation experiments, cells were incubated for 4 h with various antioxidants [including ascorbate, glutathione (GSH), butylated hydroxytoluene (BHT), beta-nicotinamide adenine dinucleotide (NADH), or beta-nicotinamide adenine dinucleotide phosphate (NADPH)] in the absence or presence of 0.1 mM PAP. For preincubation experiments, cells were incubated for 1 h with ascorbate, GSH or NADPH. Antioxidants were removed and cells were exposed to 0 or 0.1 mM PAP for 4 h. Viability was determined 24 h following PAP exposure. LLC-PK1 cells displayed a steep concentration-response relationship for PAP; 0.1 mM PAP caused approximately 50% loss of viability. Coincubation with ascorbate, GSH and NADPH was without effect on cell viability in the absence of PAP and attenuated PAP-induced losses in viability. In contrast, NADH was ineffective in preventing PAP-induced cytotoxicity. BHT alone produced a significant loss of cell viability and was ineffective in preventing PAP cytotoxicity. Inability of NADH to prevent PAP-induced cytotoxicity was related to rapid degradation of NADH in aqueous solution. Preincubation of cells with ascorbate or GSH but not NADPH was associated with attenuation of PAP-induced cytotoxicity. These data suggest that (1) PAP is cytotoxic to LLC-PK1 cells, (2) a portion of PAP cytotoxicity is due to nonenzymatic oxidation that occurs in the incubation medium, and (3) a portion of PAP cytotoxicity is due to enzymatic or nonenzymatic oxidation that occurs within cells.     

Synthesis and biological activity of mixed ligand dioxouranium(VI) and thorium(IV) complexes

Mixed ligand complexes of dioxouranium(VI) and thorium(IV) in the proportion 1:1:1 and 1:2:1 have been synthesized using 8-hydroxyquinoline as a primary ligand and L-proline and 4-hydroxy-L-proline as secondary ligands, respectively. The metal complexes have been characterized on the basis of elemental analysis, molar conductance, magnetic, spectral and thermal studies. The molar conductance studies of the complexes in DMF at 10(-3) M concentrations indicate their non-electrolytic nature. Room temperature magnetic susceptibility measurements revealed diamagnetic nature of the complexes. Electronic absorption spectra of the complexes show intra-ligand and charge transfer transitions, respectively. The thermal analysis data of the complexes indicates the presence of a coordinated water molecule/molecules. The tube dilution method has been used to study the antibacterial activity of the complexes against the pathogenic bacteria Staphylococcus aureus and Escherichia coli. The results have been compared against those of control tetracycline, which was screened simultaneously. The complexes have been screened for in vitro cytotoxicity (IC50) studies against Ehrlich ascites cells and Dalton's lymphoma ascites cells, respectively.     

Effect of brain natriuretic peptide on cyclic GMP accumulation in a kidney epithelial cell line (LLC-PK1)

The effect of porcine brain natriuretic peptide (pBNP) on cyclic GMP accumulation was studied in the kidney epithelial cell line, LLC-PK1. The addition of pBNP to the LLC-PK1 cells produced a time- and concentration-dependent increase in cyclic GMP accumulation and this effect was equipotent to that of alpha-human atrial natriuretic peptide (alpha-hANP). The simultaneous addition of pBNP and alpha-hANP at the maximal effective concentration of 10(-6) M did not have an additive effect on the cyclic GMP contents. The findings suggest that pBNP and alpha-hANP may share the same receptor in the LLC-PK1 cells.     

Role of hydrogen peroxide in cyclosporine-induced renal tubular cell (LLC-PK1) injury

We examined the role of hydrogen peroxide production in cyclosporine A (CsA)-induced LLC-PK1 injury. After exposure to CsA (0.1 microM - 100 microM), cytotoxicity assessed by lactate dehydrogenase release to the media increased dose-dependently. LLC-PK1 cells produced hydrogen peroxide, visualized by 2,7-dichlorodihydrofluorescein assay by the treatment with 100 microM CsA, that was blocked by the treatment with catalase. The cytotoxicity of CsA significantly decreased either by the treatment with catalase, mannitol, or deferoxamine, but not with superoxide dismutase. These results suggest the role of hydrogen peroxide as the source of hydroxyl radical, which mainly contributes to CsA-induced LLC-PK1 injury.     

Cytotoxic and phenotypic effects of uranium and lead on osteoblastic cells are highly dependent on metal speciation

Bone is one of the main retention organs for uranium (U) and lead (Pb). The clinical effects of U or Pb poisoning are well known: acute and chronic intoxications impair bone formation. However, only few studies dealt with the cellular and molecular mechanisms of their toxicity. The purpose of this study was to investigate acute cytotoxicity of U and Pb and their phenotypic effects on rat and human osteoblasts, the cells responsible for bone formation. The most likely species of the toxicants in contact with cells after blood contamination were selected for cell exposure. Results showed that the cytotoxic effect of U and Pb is highly dependent on their speciation. Thus, Pb was cytotoxic when left free in the exposure medium or when complexed with carbonate, cysteine or citrate, but not when complexed with albumin or phosphate, under an insoluble form. U was cytotoxic whatever its speciation, but differences in sensitivity were observed as a function of speciation. Population growth recovery could be obtained after exposure to low doses of U or Pb, except for some U-carbonate complexes which had irreversible effects whatever the dose. The activation of two markers of bone formation and mineralization, osteocalcin and bone sialoprotein (BSP), was observed after exposure to non-toxic doses or non-toxic species of U or Pb while their inhibition was observed after toxic exposure to both metals. This work provides new elements to better understand the complex mechanisms of U and Pb toxicity to osteoblasts. Our results also illustrate the importance of a strictly controlled speciation of the metals in toxicological studies.     

FIA of sildenafil citrate using UV-detection

A flow injection analysis (FIA) of sildenafil citrate (SLD) using UV detection is described in this study. The best solvent system was found to be consisting of 0.2 M phosphate buffer at pH 8 having 10% MeOH. A flow rate of 1 ml. min(-1) was pumped and active material was detected at 292 nm. The calibration equation was linear in the range of 1x10(-6)-5x10(-6) M. Limit of detection (LOD) and limit of quantitation (LOQ) were calculated to be 3x10(-7) and 8.9x10(-7) M with a R.S.D. 1.9 and 0.6% (n=7), respectively. The proposed method was applied to the determination of SLD in VIAGRA tablet, containing 50 mg active material. The results were compared with those obtained from UV-Spectrophotometry. The results showed that there is a good agreement between FIA method and the UV-Spectrophotometry. The validation studies were realised by the related applications and the results were evaluated statistically. According to the results, insignificant difference was observed between the methods.     

Role of endogenous sulfur-containing nucleophiles in an in vitro model of cis-diamminedichloroplatinum(II)-induced nephrotoxicity

Quiescent LLC-PK1 cells have been shown to be a good model of cis-diamminedichloroplatinum(II) (DDP)-induced nephrotoxicity. In these nonproliferating porcine kidney epithelial cells, DDP inhibition of protein synthesis rate is the major correlate of cytotoxicity. We report here the use of this cell line to investigate the role of endogenous sulfur-containing nucleophiles in DDP-induced nephrotoxicity. Reaction of DDP with glutathione (GSH), cysteine, or methionine for up to 24 hr led to concentration- and time-dependent loss of its toxic effects, whereas dissolution of DDP alone did not alter its reduction of viability or protein synthesis rate in LLC-PK1 cells. Treatment of these cells with differing cytotoxic concentration of DDP produced an identical transient increase in intracellular GSH, whereas compounds known to bind GSH, trans-diamminedichloroplatinum(II) (t-DDP) and diethyl maleate (DEM), rapidly depleted LLC-PK1 intracellular GSH levels. Buthionine sulfoximine (BSO) treatment decreased intracellular GSH to 10% of control without altering cell viability or protein synthesis rate. However, BSO-pretreated LLC-PK1 cells exhibited enhanced DDP-induced toxicity. CdCl2 treatment produced a 30-fold induction of metallothionein-like, cadmium-binding proteins and a 10-fold increase in metallothionein isoform I (MT-I) mRNA, but this induction had no effect on DDP-induced reduction of viability or protein synthesis rate. Protracted DDP exposure did not induce MT-I mRNA levels in LLC-PK1 cells.     

Role of P-glycoprotein in accumulation and cytotoxicity of amrubicin and amrubicinol in MDR1 gene-transfected LLC-PK1 cells and human A549 lung adenocarcinoma cells

Amrubicin is a completely synthetic 9-aminoanthracycline agent for the treatment of lung cancer in Japan. The cytotoxicity of C-13 hydroxy metabolite, amrubicinol, is 10 to 100 times greater than that of amrubicin. The transporters responsible for the intracellular pharmacokinetics of amrubicin and amrubicinol remains unclear. This study was aimed to determine whether P-glycoprotein (P-gp) plays functional and preventive role in cellular accumulation and cytotoxicity of amrubicin and its active metabolite amrubicinol by in vitro transport and toxicity experiments. Cytotoxicity and intracellular accumulation of amrubicin and amrubicinol were evaluated by LLC-PK1 cells, MDR1 gene-transfected LLC-PK1 (L-MDR1) cells overexpressing P-gp, and human A549 lung adenocarcinoma cells. L-MDR1 cells showed 6- and 12-fold greater resistance to amrubicin and amrubicinol, respectively, than the parental LLC-PK1 cells. The intracellular accumulation of both drugs in L-MDR1 cells was significantly reduced compared to the LLC-PK1 cells. The basal-to-apical transepithelial transport of both drugs markedly exceeded, whereas the apical-to-basal transport of both drugs was significantly lower in L-MDR1 cells than LLC-PK1 cells. Cyclosporin A (CyA) restored the sensitivity, intracellular accumulation and transport activity for both drugs in L-MDR1 cells. In A549 cells, CyA significantly increased the intracellular accumulation and cytotoxicity of both drugs. These findings indicated that P-gp is responsible for cellular accumulation and cytotoxicity of both amrubicin and amrubicinol, therefore suggesting that the antitumor effect of amrubicin could be affected by the expression level of P-gp in lung cancer cells in chemotherapeutic treatments.     

Bis(tri-n-butyltin)oxide induces programmed cell death (apoptosis) in immature rat thymocytes

In order to characterise the mechanism of cytotoxicity of the immunotoxic organotin compound bis(tri-n-butyltin)oxide (TBTO) to lymphoid cells, isolated thymocytes from immature rats were exposed to TBTO (0.1–5 M) for up to 6 h. At lower TBTO concentrations (0.1 and 1 M) vital staining showed that only marginal loss of viability occured, although morphological studies demonstrated increased numbers of cells with abnormal features indicative of programmed cell death (apoptosis). These changes included nuclear chromatin condensation (which was associated with increased DNA fragmentation), cytoplasmic contraction and formation of membrane bound apoptotic bodies. When visualised by agarose gel electrophoresis, genomic DNA appeared as a series of fragments with a repeat multiple of 180–200 base pairs. Comparable morphological changes and cleavage of DNA into oligonucleosomal fragments were evident in thymocytes incubated with 10 M methyl prednisolone hemisuccinate (MPS); a glucocorticoid hormone known to induce programmed cell death in thymocytes. Marked cytotoxicity associated with degenerative changes indicative of necrosis was observed in thymocytes incubated with 5 M TBTO. These findings indicate that, at levels which are not overtly cytotoxic, TBTO is capable of inducing programmed cell death in rat thymocytes. This suggest a possible mechanism for the T-cell immunodeficiency previously reported for TBTO in vivo.     

Biochemical pharmacology of N-acetyl-N-(methylcarbamoyloxy)-N'-methylurea (caracemide; NSC-253272)

Preclinical pharmacologic studies of caracemide [N-acetyl-N-(methylcarbamoyloxy)-N'-methylurea; CAR] have demonstrated a marked instability of this compound in the presence of either phosphate buffer (pH 7.4) or human plasma. Using [1-14C-acetyl]CAR and [3H-methylcarbamoyloxy]CAR, three CAR degradation products were identified: product A, N-(methylcarbamoyloxy)acetamide; product B: N-(methylcarbamoyloxy)-N'-methylurea; and product C: N-hydroxy-N'-methylurea. CAR degradation in human plasma was demonstrated by high-performance liquid chromatography (HPLC) to occur in a time- and temperature-dependent manner. A 30-min incubation (37 degrees) of CAR (10(-4) M) with human plasma resulted in degradation of more than 55% of parent compound; at 1 hr, more than 75% of original CAR was degraded. Incubation of [1-14C-acetyl]CAR with rat brain homogenate resulted in the formation of 14CO2. This reaction was partially inhibited by coincubation with physostigmine (10(-3) M). CAR inhibited acetylcholinesterase activity in neuroblastoma cells with an IC50 of 14 microM. In mechanism of action studies, CAR was found to inhibit ribonucleotide reductase activity but only at nine times the IC50 of hydroxyurea. In contrast to hydroxyurea, CAR was found to be non-cell-cycle phase-specific and non-cross-resistant with two CHO cell lines resistant to hydroxyurea. These data demonstrate the instability of CAR; moreover, they suggest that its mechanism of cytotoxicity is distinctly different from that of hydroxyurea and that the neurotoxicity associated with CAR administration may be caused in part by inhibition of acetylcholinesterase activity.     

Role of cyclic GMP on inhibition by nitric oxide donors of human eosinophil chemotaxis in vitro

1. This study was designed to investigate the effects of the nitric oxide (NO) donors sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) on N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP, 1 x 10(-7) M)-induced human eosinophil chemotaxis, cyclic guanosine-3',5'-monophosphate (cGMP) levels, protein nitration and cytotoxicity. 2. Human eosinophils were exposed to SNP, SIN-1 and SNAP (0.001-1.0 mM) for either short (10 min) or prolonged (90 min) time periods. Exposition of eosinophils with these NO donors significantly inhibited the eosinophil chemotaxis irrespective of whether cells were exposed to these agents for 10 or 90 min. No marked differences were detected among them regarding the profile of chemotaxis inhibition. 3. Exposition of eosinophils to SNP, SIN-1 and SNAP (0.001-1.0 mM) markedly elevated the cGMP levels above basal levels, but the 90-min exposition resulted in significantly higher levels compared with the 10-min protocols (5.3+/-0.6 and 2.6+/-0.2 nM 1.5 x 10(6) cells(-1), respectively). The cGMP levels achieved with SNAP were greater than SNP and SIN-1. 4. The NO donors did not induce cell toxicity in any experimental condition used. Additionally, eosinophils exposed to SNP, SIN-1 and SNAP (1.0 mM each) either for 10 or 90 min did not show any tyrosine nitration in conditions where a strong nitration of bovine serum albumin was observed. 5. Our findings show that inhibitory effects of fMLP-induced human eosinophil chemotaxis by NO donors at short or prolonged exposition time were accompanied by significant elevations of cGMP levels. However, additional elevations of cGMP levels do not change the functional profile (chemotaxis inhibition) of stimulated eosinophils.