Andrea Guidarelli

Peroxynitrite-mediated release of arachidonic acid from PC12 cells.

A short term exposure of PC12 cells to a concentration of tert‐butylhydroperoxide (tB‐OOH) causing peroxynitrite‐dependent DNA damage and cytotoxiticity promoted a release of arachidonic acid (AA) that was sensitive to phospholipase A2 (PLA2) inhibitors and insensitive to phospholipase C or diacylglycerol lipase inhibitors. The extent of AA release was also mitigated by nitric oxide synthase (NOS) inhibitors and peroxynitrite scavengers. Low levels (10u2003μM) of authentic peroxynitrite restored the release of AA mediated by tB‐OOH in NOS‐inhibited cells whereas concentrations of peroxynitrite of 20u2003μM, or higher, effectively stimulated a PLA2 inhibitor‐sensitive release of AA also in the absence of additional treatments. These results are consistent with the possibility that endogenous as well as exogenous peroxynitrite promotes activation of PLA2.

Cross-resistance to heavy metals in hydrogen peroxide-resistant CHO cell variants

Hydrogen peroxide-resistant Chinese hamster ovary (CHO) cells displayed cross-resistance to CdCl2, HgCl2 and NaAsO2 but not to Na2Cr2O7, ZnCl2, NiCl2 and CuSO4. Resistance to hydrogen peroxide and to the metals was partially retained by these cells for many generations despite growth in drug-free medium. The loss of resistance was a slow process, and was different for the various metal compounds. Cell variants had a slightly higher content of non-protein intracellular thiols (NPSH) than sensitive cells. This biochemical feature did not seem to be the cause of resistance to CdCl2 but accounted for at least part of the resistance to HgCl2 and NaAsO2. Increased metallothionein synthesis did not seem to be responsible for the metal-resistant phenotype. These results suggest that resistance to specific metal compounds in cultured mammalian cells adapted to hydrogen peroxide is dependent on a number of factors which differ for the various metal compounds and which are characterized by a different stability.

Sodium-dependent transport of ascorbic acid in U937 cell mitochondria.

U937 cells exposed to physiological concentrations of ascorbic acid (AA) accumulate the reduced form of the vitamin in the cytosol and even further in their mitochondria. In both circumstances, uptake was dependent on Na+‐AA‐cotransport, with hardly any contribution of hexose transporters, which might be recruited to transport the oxidized form of the vitamin. There was an identical linear relationship between the mitochondrial accumulation of the vitamin and the extramitochondrial AA concentration, regardless of whether detected in experiments using intact cells or isolated mitochondria. Western blot experiments revealed expression of both SVCT1 and 2 in plasma membranes, whereas SVCT2 was the only form of the transporter expressed at appreciable amounts in mitochondria. These results therefore provide the novel demonstration of SVCT2‐dependent mitochondrial transport of AA and hence challenge the present view that mitochondria only take up the oxidized form of the vitamin.

Opposite effects of nitric oxide donors on DNA single strand breakage and cytotoxicity caused by tert-butylhydroperoxide

The effects of three different NO donors on tert‐butylhydroperoxide (tB‐OOH)‐induced DNA cleavage and toxicity were investigated in U937 cells. Treatment with S‐nitroso‐N‐acetyl‐penicillamine (SNAP, 1–30 μm), while not in itself DNA‐damaging, potentiated the DNA strand scission induced by 200 μm tB‐OOH in a concentration‐dependent fashion. The enhancing effects of SNAP were observed with two different techniques for the assessment of DNA damage. Decomposed SNAP was inactive. S‐nitrosoglutathione (GSNO, 300 μm) and (Z)‐1‐[(2‐aminoethyl)‐N‐(2‐ammonioethyl) amino]diazen‐1‐ium‐1,2‐diolate (DETA‐NO, 1 mm) also increased DNA cleavage generated by tB‐OOH and these responses, as well as that mediated by SNAP, were prevented by the NO scavenger 2‐phenyl‐4,4,5,5‐tetramethylimidazolin‐1‐oxyl‐3‐oxide (PTIO). SNAP neither inhibited catalase activity nor increased the formation of DNA lesions in cells exposed to H2O2. Furthermore, SNAP did not affect the rate of rejoining of the DNA single strand breaks generated by tB‐OOH. Under the conditions utilized in the DNA damage experiments, treatment with tB‐OOH alone or associated with SNAP did not cause cell death. However, SNAP as well as GSNO markedly reduced the lethal response promoted by millimolar concentrations of tB‐OOH and these effects were abolished by PTIO. Decomposed SNAP was inactive. It is concluded that low levels of NO donors, which probably release physiological concentrations of NO, enhance the accumulation of DNA single strand breaks in U937 cells exposed to tB‐OOH. This NO‐mediated effect appears to (a) not depend on inhibition of either DNA repair (which would increase the net accumulation of DNA lesions by preventing DNA single strand break removal) or catalase activity (which would also enhance the net accumulation of DNA lesions since H2O2 is one of the species mediating the tB‐OOH‐induced DNA cleavage) and (b) be caused by enforced formation of tB‐OOH‐derived DNA‐damaging species. In contrast to these results, similar concentrations of NO prevented cell death caused by millimolar concentrations of tB‐OOH. Hence, DNA single strand breakage generated by tB‐OOH in the absence or presence of NO does not represent a lethal event.

Characterization of two novel SETX mutations in AOA2 patients reveals aspects of the pathophysiological role of senataxin

Ataxia with oculomotor apraxia (AOA) type 2 (AOA2 MIM 606002) is a recessive subtype of AOA characterized by cerebellar atrophy, oculomotor apraxia, early loss of reflexes, and peripheral neuropathy. Various mutations either in homozygous or compound heterozygous condition were so far identified in the associated gene SETX (MIM 608465). SETX encodes a large protein called senataxin with a DNA-RNA helicase domain and a putative N-terminus protein interaction domain. Here, we report the identification of two novel homozygous mutations in SETX gene, c.340_342delCTT (p.L114Del) and c.1669Cu2009>u2009T (p.R557X), in two AOA2 families. The characterization of the mutant lymphoblastoid cell lines for sensitivity to oxidative DNA-damaging agents indicates that the p.L114Del deletion confers an increased sensitivity to H2O2, camptothecin, and mitomycin C, previously found to induce death in lymphoblasts harbouring other SETX mutations; the cells carrying the nonsense mutation display instead values within the normal range. Further analysis of a neuronal cell model SKNBE, transfected with the mutant senataxin proteins, reveals increased sensitivity also to staurosporine and excitotoxicity associated with the p.L114Del mutant only. We also demonstrate that the sensitizing effect of p.L114Del on apoptosis can be reversed by senataxin silencing. The ability of a single amino acid deletion to sensitize cells to death by different agents, compared to the lack of effect of a whole protein deletion, seems to exclude a protective role played by the native protein while suggesting that a specific mutation confers to the protein the ability to enhance the toxic effect of various cell damaging agents.

Endogenous and exogenous nitric oxide enhance the DNA strand scission induced by tert-butylhydroperoxide in PC12 cells via peroxynitrite-dependent and independent mechanisms, respectively

A short‐term exposure to tert‐butylhydroperoxide (tB‐OOH) promoted a concentration‐dependent formation of DNA single‐strand breaks in PC12 cells. These events were paralleled by an increase in the cytosolic concentration of Ca2+ that was in part cleared by the mitochondria. Unlike the extent of Ca2+ mobilization and/or mitochondrial Ca2+ clearance, the DNA strand scission evoked by the hydroperoxide was markedly reduced by the nitric oxide (NO) scavenger 2‐phenyl‐4,4,5,5‐tetramethylimidazolin‐1‐oxyl‐3‐oxide (PTIO) or by the NO synthase inhibitor N‐nitro‐l‐arginine methylester (L‐NAME). Inhibitors of electron transport (rotenone and myxothiazol), ruthenium red (RR, a polycation which inhibits the calcium uniporter of mitochondria), or peroxynitrite scavengers (Trolox and l‐methionine) were as effective as PTIO or L‐NAME in inhibiting the DNA‐damaging response mediated by tB‐OOH. Rotenone, RR or peroxynitrite scavengers did not further reduce the residual DNA cleavage observed following treatment with tB‐OOH in L‐NAME‐supplemented cells. Exogenous NO also increased the DNA damage caused by tB‐OOH in L‐NAME‐supplemented cells and this response was blunted by RR or by inhibitors of electron transport but was insensitive to peroxynitrite scavengers. We conclude that both endogenous and exogenous NO enhance the DNA cleavage generated by tB‐OOH in PC12 cells. However, only endogenous NO set the bases for an involvement of peroxynitrite in this DNA‐damaging response.

L-histidine-mediated enhancement of hydrogen peroxide-induced cytotoxicity: relationships between dna single/double strand breakage and cell killing

Results presented in this study demonstrate an association between the L-Histidine-mediated enhancement of H2O2-induced cytotoxicity and the formation of DNA double strand breakage (DSB), whereas no relationship exists between the increased cytotoxic response and DNA single strand breakage (SSB). Indeed, the higher lethality and the production of DNA DSB occurred in oxidatively-injured cells regardless of whether the exposure to L-Histidine was performed before or during challenge with the oxidant. In fact, the increased level of DNA SSB detected in cells simultaneously exposed to the oxidant and the amino acid was not observed in cells pre-treated with L-Histidine and then challenged with hydrogen peroxide. Further experiments have demonstrated an association between the kinetics of DNA DSB formation and the enhancement of the cytotoxic response. In conclusion, intracellular L-Histidine seems to mediate the formation of DNA DSB and the increased growth-inhibitory response elicited by the oxidant. In addition, these results suggest that the enhancement of DNA SSB is produced by the extracellular/plasma membrane fraction of the amino acid and not causally related to the L-Histidine-mediated increase of the growth-inhibitory response to H2O2-treated cells.

Development and characterization of hydrogen preoxide-resistant Chinese hamster overy (CHO) cell variants—II. Relationships between non-protein sulfydryl levels and the induction/stability of the oxidant-resistant phenotype

Hydrogen peroxide sensitive and resistant sublines of Chinese hamster ovary (CHO) cells were tested for their non-protein sulfhydryl (NPSH) content in an attempt to establish whether a relationship exists between resistance to growth inhibition elicited by the oxidant and the NPSH pool. Cell variants characterized by increasing levels of resistance to hydrogen peroxide displayed a significant increase in cellular NPSH (expressed on a per million cell basis). Growth of resistant cells for various lengths of time in the absence of H2O2 decreased resistance, whereas the NPSH content did not vary (at least up to 127 days of growth in peroxide-free medium). The NPSH pool returned to control levels after an additional 82 days. These changes, however, were probably related to differences in cell size/amount of total cell proteins in the sublines. Indeed, when NPSH levels were expressed on a per milligram protein basis, essentially no variations were observed in sensitive and resistant sublines. It is important to note that, even by expressing the NPSH content on a per million cell basis, no correlation was found with the degree of resistance to growth inhibition elicited by the oxidant. Further experiments have demonstrated that, under conditions of reduced NPSH content (obtained by growing the cells in the presence of a glutamylcysteine synthetase inhibitor), the cytotoxic action of hydrogen peroxide was very slightly, if at all, augmented in both wild type and resistant cells. We may therefore conclude that cellular NPSH do not afford significant protection against growth inhibition induced by hydrogen peroxide in wild type cells, and that the same lack of effect occurs in cells with an increased NPSH content and carrying the oxidant-resistant phenotype.

The mechanism of the nitric oxide-mediated enhancement of tert-butylhydroperoxide-induced DNA single strand breakage

1 Caffeine (Cf) enhances the DNA cleavage induced by tert‐butylhydroperoxide (tB‐OOH) in U937 cells via a mechanism involving Ca2+‐dependent mitochondrial formation of DNA‐damaging species ( Guidarelli et al., 1997b ). Nitric oxide (NO) is not involved in this process since U937 cells do not express the constitutive nitric oxide synthase (cNOS). 2 Treatment with the NO donors S‐nitroso‐N‐acetyl‐penicillamine (SNAP, 10u2003μM), or S‐nitrosoglutathione (GSNO, 300u2003μM), however, potentiated the DNA strand scission induced by 200u2003μM tB‐OOH. The DNA lesions generated by tB‐OOH alone, or combined with SNAP, were repaired with superimposable kinetics and were insensitive to anti‐oxidants and peroxynitrite scavengers but suppressed by iron chelators. 3 SNAP or GSNO did not cause mitochondrial Ca2+ accumulation but their enhancing effects on the tB‐OOH‐induced DNA strand scission were prevented by ruthenium red, an inhibitor of the calcium uniporter of mitochondria. Furthermore, the enhancing effects of both SNAP and GSNO were identical to and not additive with those promoted by the Ca2+‐mobilizing agents Cf or ATP. 4 The SNAP‐ or GSNO‐mediated enhancement of the tB‐OOH‐induced DNA cleavage was abolished by the respiratory chain inhibitors rotenone and myxothiazol and was not apparent in respiration‐deficient cells. 5 It is concluded that, in cells which do not express the enzyme cNOS, exogenous NO enhances the accumulation of DNA single strand breaks induced by tB‐OOH via a mechanism involving inhibition of complex III.

Calcium-dependent mitochondrial formation of species promoting strand scission of genomic DNA in U937 cells exposed to tert-butylhydroperoxide: The role of arachidonic acid

Treatment of U937 cells with a sublethal concentration of tert-butylhydroperoxide generates DNA single strand breakage in U937 cells and this response is increased by caffeine, ATP, pyruvate or antimycin A. As we previously reported (Guidarelli, Clementi, Brambilla and Cantoni, (1997) Biochem. J. 328, 801–806), the enhancing effects of antimycin A are mediated by inhibition of complex III and the ensuing formation of superoxides and hydrogen peroxide in a reaction in which ubisemiquinone serves as an electron donor. Active electron transport was required in pyruvate-supplemented cells since the increased genotoxic response occurred as a consequence of enforced mitochondrial Ca2+ accumulation, a process driven by the increased electrochemical gradient. The enhancing effects of caffeine or ATP were also the consequence of mitochondrial Ca2+ accumulation but these responses were independent on electron transport. The increased formation of DNA lesions resulting from exposure to tert-butylhydroperoxide associated with the Ca2+-mobilizing agents or the respiratory substrate was mediated by arachidonic acid generated by Ca2+-dependent activation of phospholipase A2. Melittin, a potent phospholipase A2 activator, and reagent arachidonic acid mimicked the effects of caffeine, ATP or pyruvate on the tert-butylhydroperoxide-induced DNA single strand breakage.