We introduced the Escherichia coli glycolate catabolic pathway into Arabidopsis thaliana chloroplasts to reduce the loss of fixed carbon and nitrogen that occurs in C(3) plants when phosphoglycolate, an inevitable by-product of photosynthesis, is recycled by photorespiration. Using step-wise nuclear transformation with five chloroplast-targeted bacterial genes encoding glycolate dehydrogenase, glyoxylate carboligase and tartronic semialdehyde reductase, we generated plants in which chloroplastic glycolate is converted directly to glycerate. This reduces, but does not eliminate, flux of photorespiratory metabolites through peroxisomes and mitochondria. Transgenic plants grew faster, produced more shoot and root biomass, and contained more soluble sugars, reflecting reduced photorespiration and enhanced photosynthesis that correlated with an increased chloroplastic CO(2) concentration in the vicinity of ribulose-1,5-bisphosphate carboxylase/oxygenase. These effects are evident after overexpression of the three subunits of glycolate dehydrogenase, but enhanced by introducing the complete bacterial glycolate catabolic pathway. Diverting chloroplastic glycolate from photorespiration may improve the productivity of crops with C(3) photosynthesis.

Ifosfamide (IF) and cyclophosphamide (CP) are highly effective alkylating cytostatic drugs. IF and CP have to be activated through a metabolic step in vivo; numerous metabolites are known. While both IF and its structural isomer CP have severe urotoxic side effects, only IF is also a nephrotoxic drug, causing tubular damage resulting in Fanconi syndrome in some cases. Little information is available regarding the pathogenic mechanism of tubular damage by IF. We used the renal epithelial cell line LLC-PK1, which has many properties of the proximal tubule, in order to investigate the toxicity of IF and CP and of their reactive metabolites 4-hydroxy-IF (4-OH-IF), 4-hydroxy-CP (4-OH-CP), acrolein and chloroacetaldehyde (CAA). Protein content of monolayers, DNA and RNA synthesis were determined by standard techniques (thymidine and uridine incorporation). IF and CP had the lowest toxicities of all compounds tested. Both drugs inhibited thymidine incorporation by about 30% at a concentration of 300 mumol/l after 1 h incubation. 4-OH-IF and 4-OH-CP were significantly more toxic than the parent drugs. Thymidine incorporation, the most sensitive parameter, was reduced by about 70% by 300 mumol/l of either compound. In addition, 4-OH-CP reduced the total protein content of monolayers. 4-OH-IF did not effect protein content and RNA synthesis. Acrolein, the most toxic metabolite tested, reduced all three parameters significantly at concentrations of 50-75 mumol/l after 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Ifosfamide (IF) is an alkylating cytostatic with urotoxic (haemorrhagic cystitis) and nephrotoxic (Fanconi syndrome) side effects. Cyclophosphamide (CP), a structural isomer of IF, shows urotoxic but no nephrotoxic side effects. The development of haemorrhagic cystitis during therapy with IF or CP can be prevented by the uroprotective drug sodium-2-mercaptoethanesulphonate (MESNA). However, even in the presence of MESNA, Fanconi syndrome may still develop after therapy with IF. Using the renal tubular cell line LLC-PK1, we investigated whether there is a protective effect of either MESNA or of its major metabolite DIMESNA, in combination with metabolites of IF or CP, on thymidine incorporation, uridine incorporation or total protein. DIMESNA, the dimer of MESNA, is the dominant form of the molecule in the circulation; the proximal tubular cell must convert this back to MESNA at the expense of glutathione, before it can exert its uroprotective action. We did not find a protective effect of DIMESNA under any of the experimental conditions tested. LLC-PK1 cells exposed to 3 mmol/l DIMESNA did not convert DIMESNA to MESNA. The toxic effect of the CP metabolite 4-OOH-CP was more pronounced in the presence of DIMESNA than in its absence. MESNA completely prevented the toxic effects of acrolein and of 4-OOH-CP. The toxic effects of 4-OOH-IF and of chloracetaldehyde, two major metabolites of IF, were significantly reduced in the presence of MESNA. However, even at 30-fold molar excess of MESNA over a 4-OOH_IF, thymidine incorporation remained reduced by 40% compared with controls, indicating incomplete protection of tubular cells against metabolites of IF. Similarly, the effect of chloracetaldehyde was not completely reversed by MESNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Ifosfamide (IF) is an alkylating cytostatic derived from nitrogen mustard. In addition to its well-known urotoxic effects (hemorrhagic cystitis), several cases of Fanconi syndrome following IF therapy have been reported. No information is available concerning the pathomechanisms of this tubulotoxicity. We used the permanent renal epithelial cell line LLC-PK₁ in order to investigate whether major metabolites of IF (i.e. 4-OH-IF, acrolein and chloracetyldehyde) induce the transport defects most frequently detected after IF therapy in vivo. LLC-PK₁ cells of passages 162-177, grown in plastic culture dishes, were used in a confluent state. Sodium-dependent and independent fluxes of l-[³H]alanine and of D-[³H] glucose were determined by standard techniques. Activities of marker enzymes of apical and basolateral membranes, of mitochondria and of endoplasmic reticulum were determined in cell homogenates. IF itself has no detectable effect on fluxes of /-alanine and D-glucose in LLC-PK, cells. The IF metabolite 4-OOH-IF induces a clear inhibition of sodium-dependent fluxes of both substrates after a 24-hour exposure of cells to 100 µmol/l of 4-OOH-IF. Chloracetaldehyde induces a biphasic response of sodium-dependent fluxes of l-alanine with increased uptake rates at low concentrations ( < 200 µmol/l) and with a short incubation time, while higher concentrations and long exposure of the cells leads to a reduction in sodium coupled transport. Glucose transport is affected in a comparable way, however, in contrast to alanine transport, chloracetaldehyde also stimulates sodium-independent fluxes of glucose. Acrolein is the most toxic substance tested. It severely damages cell monolayers at concentrations beyond 75 µmol/l. Sodium-coupled glucose and alanine transport is inhibited by acrolein at concentrations higher than 50 µmol/l. Sodium-coupled glucose transport is more sensitive to all metabolites tested than alanine transport. While acrolein strongly affects both transport systems, marker enzymes of the apical plasma membrane, i.e. alkaline phosphatase and leucine aminopeptidase, are not significantly inhibited, suggesting a specificity of the toxic effect for the transport proteins. We conclude that LLC-PK₁ cells represent a good model for further investigation of the pathogenesis of Fanconi syndrome after IF therapy. Sodium-dependent transport systems are more sensitive to acrolein than other cell surface proteins.

Ifosfamide (IF) is an alkylating cytostatic drug with urotoxic (hemorrhagic cystitis) and nephrotoxic side effects. Several cases of Fanconi syndrome in children following therapy with IF were reported. Little information is available concerning the pathomechanisms of transport inhibition by IF. We used a permanent renal epithelial cell line with proximal tubular characteristics (LLC-PK₁) in order to investigate the effects of IF and some of its major metabolites (4-OH-IF, chloracetaldehyde, and acrolein). LLC-PK₁ cells were used in a confluent state. Sodium-dependent and sodium-independent fluxes of ³²PO₄ were determined by standard techniques. Activities of marker enzymes of apical and basolateral membranes, of mitochondria, and of endoplasmic reticulum were determined in cell homogenates. IF induces a moderate stimulation of PO₄ transport. 4-OH-IF also has a stimulatory effect on transport at low concentrations (up to 200 µmol/ l) and with short incubation (2h), while a 24-hour exposure of cells to 100 µmol/l of 4-OH-IF has an inhibitory effect of PO₄ transport. Concentrations of 4-OH-IF which inhibit transport also reduce the activity of Na⁺-K⁺-ATPase. Chloracetaldehyde, like 4-OH-IF, induces a biphasic response of PO₄ transport with stimulation in the low concentration range (up to 75 µmol/l) and inhibition at higher concentrations. Chloracetaldehyde reduces the activity of succinate-cytochrome coxidoreductase, suggesting that a defect in ATP generation might play a role in the pathogenesis of Fanconi syndrome induced by IF. Acrolein strongly damages monolayers and reduces sodium-dependent transport of P0₄ to very low levels at 150 µmol/l. It reduces the activities of both Na⁺-K⁺ ATPase and succinate-cytochrome c oxidoreductase. Acrolein also is the only metabolite with a moderate effect on alkaline phosphatase. We conclude that sodium-dependent transport of PO₄ is highly sensitive to IF metabolites. In addition to direct toxic effects of IF metabolites on transport proteins within the apical plasma membrane, damage to mitochondrial enzymes and to Na⁺-K⁺ ATPase which generates the electrochemical gradients for secondary active PO₄ transport may play an important role in the pathogenesis of Fanconi syndrome induced by IF.

Ifosfamide (IF) is an alkylating cytostatic with urotoxic and tubulotoxic side effects which may result in the development of Fanconi syndrome in children. While the urotoxicity of IF is effectively prevented by the uroprotective thiol compound sodium-2-mercaptoethanesulfonate (Mesna), tubulotoxicity of IF may occur even in the presence of Mesna and in the absence of any signs of urotoxicity. Using the renal tubular cell line LLC-PK₁, we investigated whether there is a protective effect of Mesna or of its major dimeric metabolite Dimesna against metabolites of IF with respect to the Na/H exchanger activity. We tested the major IF metabolites 4-hydroperoxy-IF (4-OOH-IF), chloroacetaldehydc (CAA), and acrolein. All metabolites significantly inhibit the Na/H exchanger activity. Half-maximal inhibition of transport occurs at concentrations of 120 µmol/l (4-OOH-IF), 80 (CAA), and 60 µmol/l (acrolein) after 2 h of incubation. The onset of the inhibitory effect of all three metabolites is rapid. Complete inhibition of Na/H exchange by acrolein and CAA is present after a 6-hour exposure to 100 µmol/l of the respective metabolite, while 100 µmol/l 4-OOH-IF causes only 50% inhibition after 24 h of incubation. Dimesna, which the proximal tubular cell has to reduce to Mesna at the expense of intracellular glutathione before it exerts a uroprotective effect, has no protective effect in LLC-PK₁ cells. Dimesna (0.3 mmol/l) displaces the dose-response curve for acrolein to the left, indicating an increased toxicity of the combination of acrolein plus Dimesna. Mesna (0.3 mmol/l) has a complete protective effect with respect to acrolein and CAA, while the protective effect versus 100 µmol/l of 4-OOH-IF is incomplete. We conclude that the function of the Na/H exchanger in LLC-PK₁ cells is altered by metabolites of IF. The incomplete protection against the toxic effect of 4-OOH-IF by Mesna may explain the pathomechanism by which IF causes tubulotoxicity in the absence of urotoxicity.

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It is known, that the makroheteropolyanion [Sb8W20O80]16- protects mice against Friend Leukemia. We found the similar effect of two other great heterpolytungstates and the in vivo inactivity of some smaler related compounds. Some aspects of the mode of action of these substances are discussed.