Inhibition by spermine of the induction of nitric oxide synthase in J774.2 macrophages: requirement of a serum factor.

Polyamines are endogenous regulators of various cell functions. Nitric oxide (NO) is a cytostatic and cytotoxic free radical which is produced by the inducible NO synthase (iNOS) in immuno-stimulated macrophages. We tested whether spermine modulates the induction of iNOS in J774.2 macrophages. Stimulation of macrophages by bacterial lipopolysaccharide (LPS) or gamma-interferon increased the accumulation of nitrite in the culture medium. Spermine (10(-6) - 10(-4) M) inhibited nitrite production without causing cytotoxicity. This inhibition of NO formation by spermine was significantly reduced when it was given 6 h after LPS. Spermine did not inhibit nitrite accumulation when foetal calf serum was omitted from the tissue culture medium. Thus, spermine is an inhibitor of the induction of iNOS, and its inhibitory activity requires the presence of a serum factor.     

Reduced glutathione levels and expression of the enzymes of glutathione synthesis in cryopreserved hepatocyte monolayer cultures.

Cryopreservation of monolayers of hepatocytes in a freezing medium containing 10% (v/v) dimethylsulfoxide, 90% (v/v) foetal calf serum retains cell morphology and viability, but cells lose up to 50% of their intracellular reduced glutathione. This is accompanied by a small increase in glutamate cysteine ligase expression in cryopreserved cultures, but glutathione synthetase expression is undetectable post-cryopreservation. Inclusion of ascorbic acid and alpha-tocopherol in the freezing medium improves maintenance of reduced glutathione content post-cryopreservation at 84% of the levels in non-cryopreserved monolayer cultures, but does not restore glutathione synthetase expression. The inability to synthesise reduced glutathione will mean that cryopreserved hepatocyte monolayers are more susceptible to toxic insults.     

Spermine deficiency resulting from targeted disruption of the spermine synthase gene in embryonic stem cells leads to enhanced sensitivity to antiproliferative drugs

Polyamines are known to be essential for normal cell growth and differentiation. However, despite numerous studies, specific cellular functions of polyamines in general and individual polyamines in particular have remained only tentative, because of a lack of appropriate cell lines in which genes of polyamine-synthesizing enzymes have been disrupted by gene targeting. With the use of homologous recombination technique, we disrupted the gene encoding spermine synthase in mouse embryonic stem cells. The spermine synthase gene is located on X chromosome in mouse and, because the cells used in this study were of XY karyotype, a single targeting event was sufficient to result in null genotype. The targeted cells did not have any measurable spermine synthase activity and were totally devoid of the polyamine spermine. Spermine deficiency led to a substantial increase in spermidine content, but the total polyamine content was nearly unchanged. Despite the lack of spermine, these cells displayed a growth rate that was nearly similar to that of the parental cells and showed no overt morphological changes. However, the spermine-deficient cells were significantly more sensitive to the growth inhibition exerted by 2-difluoromethylornithine, an inhibitor of ornithine decarboxylase. Similarly, methylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, and diethylnorspermine, a polyamine analog, although exerting cytostatic growth inhibition on wild-type cells, were clearly cytotoxic to the spermine-deficient cells. The spermine-deficient cells were also much more sensitive to etoposide-induced DNA damage than their wild-type counterparts.     

Monovalent cation (MC) current in cardiac and smooth muscle cells: regulation by intracellular Mg2+ and inhibition by polycations

1 Previously we have described a monovalent cation (MC) current that could be unmasked by the removal of extracellular divalent cations in vascular smooth muscle cells (SMC) and cardiac myocytes, but specific and potent inhibitors of MC current have not been found, and the mechanism of its intracellular regulation remains obscure. 2 Here we show that small MC current is present in intact cells and could be dramatically up-regulated during cell dialysis. MC current in dialyzed cells strongly resembled monovalent cation current attributed to Ca(2+) release-activated Ca(2+)-selective (CRAC) channels, but its activation did not require depletion of Ca(2+) stores, and was observed when the cells were dialyzed with, or without BAPTA. 3 Intracellular free Mg(2+) inhibits MC current with K(d)=250 microM. 4 Extracellular (but not intracellular) spermine effectively blocked MC current with K(d) =3-10 microM, while store-operated cations (SOC) channels and capacitative Ca(2+) influx were not affected. 5 Spermine effectively inhibited MC current-induced SMC depolarization, and prevented Ca(2+) paradox-induced vascular contracture. 6 Both, MC and SOC currents were inhibited by 2-aminoethoxydiphenyl borate (2-APB). 7 It is concluded that MC current could be regulated by intracellular Mg(2+), and low concentrations of extracellular spermine could be used to discriminate it from SOC current, and to assess its role in cellular function.     

Alterations on polyamine content and glutathione metabolism induced by different concentrations of paraquat in CHO-K1 cells.

The effect of herbicide paraquat has been assessed on CHO-K1 cultures at different concentrations. Glutathione peroxidase, reductase and S-transferase, as well as total and oxidized glutathione, were evaluated along the standard growth curve of the cultures. Paraquat was then administered during mid-log phase at concentrations that produced a calculated lethality of 6.25%, 12.5% and 25%, using the lysosomal dye assay, neutral red. After 24hr of incubation with paraquat, glutathione peroxidase suffered a large dose-response increase, unlike glutathione reductase and S-transferase, the activities of which were lower than untreated controls. The profile of total glutathione content was similar to that found for glutathione peroxidase, increasing with the administered doses of the herbicide. Polyamine content has been also studied at the same concentrations of paraquat, showing that intracellular spermidine and spermine pools were negatively affected with paraquat in a dose-response manner, unlike putrescine, which maintained elevated pools at the three concentrations assayed.     

Epigallocatechin-3-gallate(-)protects Chang liver cells against ethanol-induced cytotoxicity and apoptosis

The objective of the study was to investigate the effect of epigallocatechin-3-gallate (EGCG) on ethanol (EtOH)-induced cytotoxicity in human Chang liver cells. Cells were incubated with either 30 mM EtOH alone or together in presence of (25 microM) EGCG for 24 hr. Assays were performed in treated cells to evaluate the ability of EGCG to prevent the toxic effects of EtOH. EtOH exposure suppressed the growth of Chang liver cells and induced lactate dehydrogenase leakage, oxygen radical formation, peroxidation of lipids, mitochondrial dysfunction and apoptosis. Reduced glutathione (GSH) concentration was significantly decreased (P < 0.05) while oxidized glutathione (GSSG) concentration was significantly elevated in EtOH-treated cells as compared to normal cells. Incubation of EGCG along with EtOH significantly prevented EtOH-dependent cell loss and lactate dehydrogenase leakage. This was associated with a reduction in oxidative damage as reflected by a reduction in the generation of reactive oxygen species, and in lipid peroxidation and maintenance of intracellular GSH/GSSG ratio. EGCG decreased the accumulation of sub-G(1) phase cells and reduced apoptosis. The findings suggest that EGCG exerts a protective action during EtOH-induced liver cell damage.     

Protective effects of glutathione on diethyldithiocarbamate (DDC) cytotoxicity: a possible mechanism

Rat cerebral astrocytes grown in culture were exposed to 35 micrograms diethyldithiocarbamate (DDC)/ml of medium for 1 hr and treated with 0 or 10 mM reduced glutathione (GSH) 1 hr post-DDC. DDC treatment resulted in a 90% reduction in cell adherence within 24 hr and complete inhibition of growth. The most pronounced ultrastructural lesion in DDC-treated cells was on mitochondria. Numerous lipofuscin-like deposits were seen in these cells. In addition, DDC treatment resulted in a greater than 400% increase in cellular copper. The activity of the selenoenzyme glutathione peroxidase was reduced by about 40% with no concomitant effect on cytosolic superoxide dismutase activity. The data suggest that DDC cytoxicity is peroxidative in nature, presumably due to the massive influx of copper into the astrocyte. GSH treatment 1 hr after exposure of the cells to DDC completely prevented the DDC-induced reduction in cell adherence and growth inhibition. Ultrastructurally, cells post-treated with GSH prevented much of the damage caused by DDC. This protection was associated with marked reduction in cellular copper and a return to control glutathione peroxidase activity.     

Inhibition of GTPase activity of Gi proteins and decreased agonist affinity at M2 muscarinic acetylcholine receptors by spermine and methoctramine.

1. The effects of spermine and methoctramine, a selective M2 muscarinic receptor antagonist, were studied on the high-affinity GTPase activity of G proteins, and on ligand binding to M2 muscarinic receptors in pig heart sarcolemma. 2. The spontaneous GTP hydrolysis by pig heart sarcolemma and its stimulation by mastoparan or carbachol were prevented by pertussis toxin and inhibited by methoctramine (IC50s: 21, 13 and 0.005 microM, respectively), and spermine (IC50s: 967, 278 and 11 microM). Spermine and methoctramine also inhibited spontaneous GTP hydrolysis by rat peritoneal mast cell membranes which do not respond to carbachol. 3. The neutral muscarinic antagonists, AF-DX 116 and atropine, did not modify the inhibitory effect of high concentrations of methoctramine, indicating that this effect was not related to the antagonist binding site of muscarinic receptors. We suggest that methoctramine behaves as a receptor antagonist at nanomolar concentrations and interacts with G proteins at micromolar concentrations. 4. Spermine did not modify the binding of the tritiated muscarinic antagonist [3H]-NMS, but decreased the binding of the agonist [3H]-Oxo-M. Spermine elicited a rightward shift of the carbachol/[3H]-NMS binding isotherm with a decrease in the proportion of sites with high-affinity for carbachol, suggesting that polyamines uncouple Gi proteins from receptors. 5. The inhibition of GTPase activity by polyamines, preventing the re-association of alpha and betagamma subunits of Gi proteins, might sustain the regulatory effect of Gi subunits on downstream effectors. The level of intracellular polyamines might be important for the control of the transduction of extracellular signals through Gi protein-coupled receptors.     

The mechanism of the inhibitory effect of polyamines on the induction of nitric oxide synthase: role of aldehyde metabolites.

1. We have recently found that in the presence, but not in the absence, of foetal calf serum, spermine inhibits the production of nitric oxide (NO) in cultured J774.2 macrophages stimulated with bacterial endotoxin (lipopolysaccharide; LPS) or with gamma-interferon (IFN), showing that polyamines may act as suppressants of NO-mediated immune functions. Here, we have studied the mechanisms and the specificity of this inhibitory action. 2. Other polyamines, as well as spermine, inhibit the formation of NO in cultured J774.2 macrophages, with the order of potency being spermine > spermidine >> putrescine = cadaverine. This inhibition of NO formation is not due to any cytotoxic effect of these agents for they neither reduced mitochondrial respiration nor increased the release of lactate dehydrogenase into the supernatant. 3. Spermine is not a direct inhibitor of the activity of iNOS in induced J774.2 cells as measured by its lack of effect on the conversion of L-arginine to L-citrulline in homogenates. Neither spermine, nor its metabolites, interfere with the production of nitrite from NO or act as scavengers of NO. Thus, spermine is an inhibitor of the induction of iNOS. 4. Spermine inhibits nitrite formation in the presence of foetal, newborn or adult bovine serum, but not rat or human serum. 5. The effect of sper mine on nitrite production can be prevented by isoniazid, hydrazine or hydroxylamine, inhibitors of spermine oxidase, as well as by phenylhydrazine, an aldehyde inhibitor. We have, therefore, tested the effects of spermine dialdehyde or malon dialdehyde on the induction of iNOS. Spermine dialdehyde (SDA, 10(-5) M) inhibits nitrite formation by IFN-activated J774.2 cells in the absence of serum when given as a pretreatment but not when given 6 h after stimulation. In contrast, malon dialdehyde was ineffective. Thus, aldehyde metabolites of spermine, such as SDA, account for the inhibitory effect of polyamines on the induction of NOS in vitro. 6. The inhibitory effect of polyamines on iNOS induction appears to be fairly specific to iNOS, for spermine does not inhibit LPS-induced production of prostaglandin F2 alpha or tumour necrosis factor.     

Glutathione enhancement in various mouse organs and protection by glutathione isopropyl ester against liver injury

Intraperitoneal administration of glutathione isopropyl ester to fasted, male NMRI mice dose dependently increased the glutathione concentration in various organs. Administration of 1 g/kg glutathione isopropyl ester led to the following increases: liver 166%; lung 164%; heart 121% after 4 hr; and brain 133% after 6 hr. Spleen, kidney, muscle, serum and blood cell glutathione were not affected by the treatment. Pretreatment with glutathione isopropyl ester was found to protect against paracetamol- or allyl alcohol-induced liver damage. Following treatment with the ester a significant correlation between protection against liver damage and enhancement of liver glutathione content was obtained. The dose dependence of this protection was studied.     

Polyamine modulation of iron uptake in CHO cells

Polyamines are ubiquitous molecules, which, like iron, are essential for cell growth. All eukaryotic cells are equipped with a specific polyamine transport system (PTS). Polyamines have primary and secondary amino groups which chelate bivalent metal cations such as Fe and Cu. In the present study, we investigated the potential contribution of naturally occurring polyamines and their active transport system to iron uptake. In presence of subtoxic Fe(III) (10microM), treatment of CHO cells with spermine, and to a lesser extent with spermidine (10-100microM), resulted in a marked cytotoxic effect. This cytotoxicity was prevented by the addition of an iron-chelator, deferioxamine, and was not observed in CHO-MG cells, a mutant cell line devoid of polyamine transport activity. Experiments using 14C-polyamines and 55Fe(III) revealed that these toxic effects were related to polyamine-modulation of iron uptake, and were dependent on the presence of the active PTS. These results demonstrated active uptake of polyamine-iron complexes via the PTS. The number of amino groups affected the efficacy of the studied natural polyamines to transport iron via the PTS. Spermine, a tetramine, was more efficient than the triamine spermidine. Co-transport of iron by the diamine putrescine was not observed. These results demonstrate that the cell polyamine transport system is a potential cell entry pathway for iron. The studied polyamines, spermine and spermidine, may be components of the pool of transferrin-independent iron-chelating vectors, which have recently attracted the attention of many investigators.     

Chromium (VI)-induced cytotoxicity to osteoblast-derived cells

Chromium is well recognized as a carcinogen, and there are concerns about local and systemic cytotoxicity and carcinogenicity during the use of chromium-containing alloys for orthopaedic implants. We have investigated the cytotoxicity of Cr VI in immortalized rat osteoblast cells in vitro using alkaline phosphatase (ALP) activity as an index of toxicity. Cr VI caused a concentration-dependent decrease in ALP activity, thought to be mediated by intracellular reduction to Cr III. The role of several antioxidant vitamins, reduced glutathione (GSH) and DT-diaphorase in the reduction of Cr VI was investigated. The toxic response to Cr VI was partially prevented by treating the cells with ascorbic acid. In contrast, riboflavin and -tocopherol did not alter the response. Ascorbic acid is thought to reduce Cr VI to Cr III extracellularly. This protects the cells because they are relatively impermeable to Cr III. Treatment of cells with dicoumarol, an inhibitor of DT-diaphorase activity, also decreased the toxicity of Cr VI, suggesting that this enzyme is involved in the intracellular reduction of the metal. GSH was not depleted during the metabolism of Cr VI and this was probably due to the activity of glutathione reductase which acts to recycle oxidized glutathione. However, depletion of intracellular GSH by buthionine sulfoximine increased the toxicity of Cr VI at early time points (after 1.5 and 3 hr exposure). GSH and Cr VI therefore interact in the osteoblasts, and this may be through formation of a conjugate and/or by detoxification of reactive intermediates formed during redox cycling of the chromium.     

Contribution of serum and cellular semicarbazide-sensitive amine oxidase to amine metabolism and cardiovascular toxicity.

Semicarbazide-sensitive amine oxidase (SSAO) plays a role in the in vivo and in vitro toxicity of several environmental and endogenous amines. We investigated the role of SSAO as a component of cell culture medium (through addition of fetal calf serum (FCS)) compared to intracellular SSAO in the in vitro cytotoxicity of three amines and metabolites. Smooth muscle cells and beating cardiac myocytes were grown in 96-well plates and exposed to various concentrations and combinations of FCS in medium, amines (allylamine, AA; benzylamine, BZA; and methylamine, MA), and amine metabolites (aldehydes: acrolein, benzaldehyde, and formaldehyde; hydrogen peroxide, H2O2; ammonia, NH3). Amine and amine metabolite cytotoxicity was quantified by monitoring cell viability. SSAO activity was measured in FCS, cardiovascular cells, or rat plasma by a radioenzymatic assay using [14C]BZA. Our data show that AA and its aldehyde metabolite, acrolein, were the most toxic compounds to both cell types. However, AA toxicity was FCS-dependent in both cell types, while BZA, MA, and amine metabolite (i.e., aldehydes, H2O2, and NH3) cytotoxicity showed little FCS dependence. In these experiments, medium containing 10% FCS had a calculated amine metabolic capacity that was 30- to 50-fold that of the cultured smooth muscle cellular content in a single well of a 96-well plate. Our study demonstrates that SSAO in FCS contributes to amine metabolism and cytotoxicity to rat cardiovascular cells in vitro and how critical it is to evaluate serum for its role in mechanisms of amine toxicity in vitro and in vivo.     

Zinc effects on glutathione metabolism relationship to zinc-induced protection from alkylating agents

Several aspects of the effects of zinc on the metabolism of glutathione were examined in the Chinese hamster cell (line CHO) and in three derived sublines which differ in their resistance to the thiol reactive heavy metal cadmium. In the parental CHO cell, which does not induce the synthesis of metallothionein in response to zinc, glutathione levels remained approximately constant during the first 6 hr of zinc exposure. In the resistant cell lines, which induce the synthesis of metallothionein in response to zinc, the glutathione levels dropped transiently during zinc exposure. In all cell lines except the most cadmium resistant line, the glutathione levels after 12 hr were increased up to 3-fold relative to pretreatment levels. Similarly, the glutathione S-transferase activity measured by the conjugation of 1-chloro-2,4-dinitrobenzene to glutathione was increased after 9-12 hr of zinc treatment in all except the most highly cadmium resistant cell line. Glutathione reductase was not affected consistently by zinc treatment; however, the level of activity of this enzyme in the most highly cadmium resistant line was two to three times greater than that observed in the other cell lines. These effects are considered in relation to the zinc-induced protection of these cells from the toxic effect of the alkylating agent melphalan.     

Mechanism of chemical-induced toxicity. II. Role of extracellular calcium

Previous studies disagree as to if chemical-induced cell death is caused by the influx and accumulation of extracellular Ca2+. To determine the role of extracellular Ca2+ in toxic cell death, the viability (leakage of intracellular K+ and lactate dehydrogenase) and total Ca2+ content of isolated hepatocytes incubated in the presence or absence of extracellular Ca2+ were determined during a toxic insult with bromobenzene, ethyl methanesulfonate (EMS), Ca2+ ionophore A23187, and adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). The present study utilized the dibutyl phthalate separation technique which enabled the analysis of only viable hepatocytes for changes in intracellular Ca2+ and K+ content during toxic cell injury. The three chemical treatments, bromobenzene, EMS, and ADR-BCNU, each caused an accelerated loss of viability in hepatocytes incubated without extracellular Ca2+ as compared to cells incubated with Ca2+. Furthermore, the total Ca2+ content of viable hepatocytes incubated in the presence of extracellular Ca2+ did not increase during chemically induced cell injury as compared to control cells. In fact, a significant decline in total cellular Ca2+ was observed in viable hepatocytes incubated in Ca2+-free medium during toxic cell injury. Treatment with Ca2+ ionophore A23187 was also toxic to hepatocytes incubated in the presence or absence of extracellular Ca2+. At high concentrations of ionophore (20 microM or 4 micrograms/10(6) cells), cell death was accelerated in hepatocytes incubated with Ca2+ as compared to cells incubated in Ca2+-free medium. In contrast, after treatment with lower concentrations of ionophore (10 microM or 2 micrograms/10(6) cells), the rate of cell death was reversed with hepatocytes incubated without extracellular Ca2+ dying first. Thus, depending on the concentration of A23187 and the time of exposure, the presence of extracellular Ca2+ can be shown either to accelerate or protect against cell death. Surprisingly, reversible and irreversible cell injury were not observed in hepatocytes incubated with extracellular Ca2+ and 2 microM A23187 though this treatment resulted in an 800% increase in total intracellular Ca2+ content. We conclude that chemical-induced hepatic cell death is not caused by an increase in total cellular Ca2+ resulting from the influx of extracellular Ca2+.     

Exogenous glutathione decreases cellular cadmium uptake and toxicity.

The effect of intracellular glutathione (GSH) on cadmium metabolism and toxicity has been extensively investigated. However, little is known regarding the effect of extracellular GSH on cellular cadmium responses. Therefore, this study was conducted to investigate the effect of exogenously added GSH on cadmium toxicity in normal rat kidney fibroblasts (NRK-49F). Exponentially growing NRK-49F cells were arrested by serum deprivation and then stimulated with epidermal growth factor (EGF). CdCl2, at concentrations that range from 0.25 to 2 microM, was found to inhibit, in a dose-dependent fashion, the EGF-induced DNA synthesis (as judged by [3H]thymidine incorporation) in the cells. A long-term survival assay revealed that CdCl2 above 1 microM was toxic to the cells. Exogenous GSH had a dose-dependent antagonistic effect on cadmium inhibition of EGF-induced DNA synthesis, and 1 mM GSH was found to block completely cadmium inhibition of both EGF-induced DNA synthesis and cell survival. Exogenously added GSH did not increase intracellular GSH levels but decreased cadmium accumulation by the cells. This decrease was primarily caused by a reduced cadmium uptake. Further studies indicated that exogenous GSH would form a complex with cadmium outside of the cells preventing cellular cadmium uptake. This may explain the mechanism of action of the exogenous GSH in cytoprotection against cadmium. The results also suggested a practical potential for GSH as a cadmium chelator. If GSH were coadministered with a cadmium mobilizer and a gamma-glutamyl transpeptidase inhibitor, it could enhance cadmium excretion from the body.     

Anethole dithiolethione, a putative neuroprotectant, increases intracellular and extracellular glutathione levels during starvation of cultured astroglial cells

Astroglial cells protect neurons against oxidative damage. The antioxidant glutathione plays a pivotal role in the neuroprotective action of astroglial cells which is impaired following loss of glutathione. Anethole dithiolethione (ADT), a sulfur-containing compound which is used in humans as a secretagogue, increases glutathione levels in cultured astroglial cells under “physiological” conditions and is thought thereby to protect against oxidative damage. Presently, we report the effect of ADT (3–100 μM) on glutathione content of and efflux from rat primary astroglia-rich cultures under “pathological” conditions, i.e., extended deprivation of glucose and amino acids. Although cellular viability was not affected significantly, starvation of these cultures for 24 h in a bicarbonate buffer lacking glucose and amino acids led to a decrease in glutathione and protein content of approximately 43% and 40%, respectively. Although no effect on the protein loss occurred, the presence of ADT during starvation counteracted the starvation-induced loss of intracellular glutathione in a concentration-dependent way. At a concentration of 100 μM ADT even a significant increase in astroglial glutathione content was noted after 24 h of starvation. Alike intracellular glutathione levels, the amount of glutathione found in the buffer was elevated substantially if ADT was present during starvation. This ADT-mediated, apparent increase in glutathione efflux was additive to the stimulatory effect on extracellular glutathione levels of acivicin (100 μM), an inhibitor of extracellular enzymatic glutathione breakdown. However, the ADT-induced elevation of both intra- and extracellular glutathione content during starvation was prevented completely by coincubation with buthionine sulfoximine (10 μM), an inhibitor of glutathione synthesis. These results demonstrate that, most likely through stimulation of glutathione synthesis, ADT enables astroglial cells to maintain higher intra- and extracellular levels of glutathione under adverse conditions. Considering the lowered glutathione levels in neurodegenerative syndromes, we conclude that further evaluation of the therapeutic potential of the compound is warranted. Received: 2 June 1998 / Accepted: 18 September 1998     

Role of apoptosis in cisplatin-induced toxicity in the renal epithelial cell line LLC-PK1. Implication of the functions of apical membranes

The role of apoptosis and the implications of the functions of apical membranes in cisplatin-induced nephrotoxicity were investigated using the kidney epithelial cell line LLC-PK1. When LLC-PK1 cells were treated with 30 microM cisplatin, the number of floating cells was increased markedly. However, the number was not increased by treatment with 1 mM cisplatin, suggesting that different mechanisms were involved in the toxicities of these two treatments. DNA fragmentation, condensation of nuclear chromatin, and the absence of trypan blue staining suggested that cellular toxicity following treatment with 30 microM cisplatin for 24 hr was mediated predominantly by apoptosis. Specific activities of apical enzymes (gamma-glutamyltransferase, EC 2.3.2.2; and alkaline phosphatase, EC 3.1.3.1) in LLC-PK1 cells were decreased markedly by treatment with 30 microM cisplatin for 24 hr, whereas neither lactate dehydrogenase (LDH; EC 1.1.1.27) release nor a decrease in cellular protein content was observed following the same treatment. In addition, concomitant treatment with reduced glutathione completely attenuated both the apoptosis and the decrease of apical enzyme activities induced by 30 microM cisplatin. Neither DNA fragmentation nor condensation of chromatin was induced by treatment with 1 mM cisplatin for 12 hr. However, LDH release and a decrease in cellular protein level were induced by 1 mM cisplatin, suggesting that the toxic effect was due to necrosis. Under these conditions, specific activities of apical enzymes were not decreased. These results suggested that apoptosis was more responsible than necrosis for the loss of apical functions in cisplatin-induced toxicity in LLC-PK1 cells.     

Deoxyribonucleoside triphosphate pools and adenosine toxicity

The toxic effect of adenosine on the metabolism of malignant lymphoid cells has been studied in relation to the activity of intracellular adenosine deaminase. Exposure in vitro of L1210 and L5178Y cells for 48 hr to adenosine demonstrated that concentrations above 10^?5 M inhibited cell division, the toxic effect being inversely proportional to intracellular adenosine deaminase levels. Measurement of the deoxyribonucleoside triphosphate pools in cells exposed to adenosine resulted in a 22 per cent reduction in the pyrimidine deoxyribonucleoside triphosphates. Adenosine-mediated growth inhibition was markedly enhanced by coformycin, a potent inhibitor of adenosine deaminase.     

Hydroxylamine-containing inhibitors of polyamine biosynthesis and impairment of colon cancer cell growth

Polyamine synthesis (by the action of ornithine decarboxylase [ODC] and S-adenosylmethionine decarboxylase [SAMDC]) and polyamine content are high in colon cancer. In addition, colonic lumen is rich in polyamines synthesised by colonic microflora; for this reason, polyamine depletion in colon cancer may be a logical approach to impair growth of colon cancer cells. We evaluated highly specific and reportedly non-toxic hydroxylamine-containing inhibitors of ODC (1-aminooxy-3-aminopropane, APA) and SAMDC (S-(5'-deoxy-5'-adenosyl)-methylthioethyl-hydroxylamine, AMA) in human colon cancer cells (Caco-2 and HT-29) in culture. APA depleted ODC activity within 24 hr, more rapidly than did difluoromethylornithine. APA and AMA in combination (100 microM each) reduced ODC and SAMDC activities to undetectable levels within 24 hr and intracellular polyamines to 8-23% of control. The resulting growth arrest could be reversed only by twice as much spermidine as is physiologically present in the colonic lumen. In concentrations sufficient to deplete growth, APA and AMA were not toxic. Simultaneous treatment with APA, AMA, and 5-fluorouracil reduced colon cancer cell survival more potently than treatment with 5-fluorouracil alone. The hydroxylamine-containing ODC and SAMDC inhibitors APA and AMA are potent inhibitors of colon cancer cell proliferation and might be therapeutically promising in colon cancer.