Mechanisms of nitrogen oxide-mediated disruption of metalloprotein function: an examination of the copper-responsive yeast transcription factor Ace1.

Nitric oxide (NO) has been found to inhibit the copper-responsive yeast transcription factor Ace1 in an oxygen-dependent manner. However, the mechanism responsible for NO-dependent inhibition of Ace1 remains unestablished. In the present study, the chemical interaction of nitrogen oxide species with Ace1 was examined using a yeast reporter system. Exposure of yeast to various nitrogen oxides, under a variety of conditions, revealed that the oxygen-dependent inhibition of Ace1 is due to the reaction of NO with O(2). The nitrosating nitrogen oxide species N(2)O(3) is likely to be the disrupter of Ace1 activity. Considering the similarity of metal-thiolate ligation in Ace1 with other mammalian metalloproteins such as metallothionein, metal chaperones, and zinc-finger proteins, these results help to understand the biochemical interactions of NO with those mammalian metalloproteins.

Effects of nitric oxide on the copper-responsive transcription factor Ace1 in Saccharomyces cerevisiae: cytotoxic and cytoprotective actions of nitric oxide.

Previous studies indicate that nitric oxide (NO) can serve as a regulator/disrupter of metal-metabolizing systems in cells and, indeed, this function may represent an important physiological and/or pathophysiological role for NO. In order to address possible mechanisms of this aspect of NO biology, the effect of NO on copper metabolism and toxicity in the yeast Saccharomyces cerevisiae was examined. Exposure of S. cerevisiae to NO resulted in an alteration of the activity of the copper-responsive transcription factor Acel. Low concentrations of the NO donor DEA/NO were found to slightly enhance copper-mediated activation of Acel. Since Acel regulates the expression of genes responsible for the protection of S. cerevisiae from metal toxicity, the effect of NO on the toxicity of copper toward S. cerevisiae was also examined. Interestingly, low concentrations of NO were also found to protect S. cerevisiae against the toxicity of copper. The effect of NO at high concentrations was, however, opposite. High concentrations of DEA/NO inhibited copper-mediated Acel activity. Correspondingly, high concentrations of DEA/NO (1 mM) dramatically enhanced copper toxicity. An intermediate concentration of DEA/NO (0.5 mM) exhibited a dual effect, enhancing toxicity at lower copper concentrations (<0.5 mM) and protecting at higher (> or =0.5 mM) copper concentrations. Thus, it is proposed that the ability of NO to both protect against (at low concentrations) and enhance (at high concentration) copper toxicity in S. cerevisiae is, at least partially, a result of its effect on Acel. The results of this study have implications for the role of NO as a mediator of metal metabolism.

The interaction of nitric oxide (NO) with the yeast transcription factor Ace1: A model system for NO-protein thiol interactions with implications to metal metabolism.

Nitric oxide (NO) was found to inhibit the copper-dependent induction of the yeast CUP1 gene. This effect is attributable to an inhibition of the copper-responsive CUP1 transcriptional activator Ace1. A mechanism is proposed whereby the metal binding thiols of Ace1 are chemically modified via NO- and O(2)-dependent chemistry, thereby diminishing the ability of Ace1 to bind and respond to copper. Moreover, it is proposed that demetallated Ace1 is proteolytically degraded in the cell, resulting in a prolonged inhibition of copper-dependent CUP1 induction. These findings indicate that NO may serve as a disrupter of yeast copper metabolism. More importantly, considering the similarity of Ace1 to other mammalian metal-binding proteins, this work lends support to the hypothesis that NO may regulate/disrupt metal homeostasis under both normal physiological and pathophysiological circumstances.

Differential changes in rat brain nitric oxide synthase in vivo and in vitro by methylmercury.

Alterations in mRNA level, protein content and enzyme activity for nitric oxide synthase (NOS) in the cerebrum and cerebellum during a continuous exposure of neurotoxic metal, methylmercury, were examined in Wistar rats. Subcutaneous (s.c.) administration of methylmercuric chloride (MMC, 10 mg kg-1 day-1, 8 days) resulted in significant increases with time of NOS activities in the cerebrum (1. 6-1.9-fold, 5-8 days) and cerebellum (1.4-fold, 8 days). RT-PCR and immunoblot analyses indicated that the increase in the enzyme activity caused by this metal appears to be due to increase in protein levels of neuronal NOS (nNOS), but not inducible NOS (iNOS) because little appreciable mRNA and protein for iNOS were seen during MMC exposure. The direct effect of mercuric compounds on nNOS activity in vitro was evaluated using 20,000xg supernatant from rat cerebellum homogenate. In contrast to the in vivo observation, inorganic-, alkyl-, and aryl-mercuric compound showed potent inhibition of nNOS activity with IC50 values of 11-43 microM, whereas dimethylmercury (DMM) was without effect on the enzyme activity. Further experiments indicated that the inhibition of nNOS by organomercurial occurred via thiol modification.

Application of synchrotron radiation X-ray fluorescence imaging combined with histochemical staining to the renal section of mercury-treated rats.

A combination method consisting of synchrotron radiation X-ray fluorescence imaging and histochemical staining was employed to examine the detailed distribution of metal elements and morphological changes in the kidney section of rats exposed simultaneously to mercurial. A single injection of mercuric chloride (5 mg kg (-1)) to rats resulted in significant urinary leakages of the biological markers for acute mercury intoxication. Under this condition (i) a striking damage of brush border, cell loss and a dominant accumulation of mercury in the proximal tubules, (ii) a good correlation between the tubular damage caused by mercury and localization of the toxic metal, and (iii) an obvious distribution difference between zinc and sulfur following mercury exposure, were successfully shown by the present procedure with a 20 micron thick specimen.

Post-transcriptional elevation of mouse brain Mn-SOD protein by mercuric chloride.

Alterations in gene expression, protein content and enzyme activity of brain Mn-SOD following mercuric chloride (HgCl2) exposure were examined in ICR male mice. Subcutaneous administration of HgCl2 (1 mg Hg/kg) resulted in a significant increase (4-fold) in the brain Mn-SOD content at 6 h after injection while the total mercury concentration was about 0.11 microg/g of brain. The enhancement of Mn-SOD protein caused by HgCl2 was completely abolished by pretreatment with dexamethasone (3 mg/kg) 1 h prior to HgCl2 administration, suggesting involvement of inflammation in inorganic mercury-induced increase in the antioxidant enzyme. This increase in level of Mn-SOD content coincided with a substantial rise in the enzyme activity; however, Northern blot analysis revealed that the induction of protein level was not due to that of its gene expression. The results of the present study indicate that mouse brain Mn-SOD appears to undergo post-translational modification by the environmental toxic metal, and induction of the antioxidant enzyme could be of an initial response to the metal-induced oxidative stress.

Mercury dynamics in hair of rats exposed to methylmercury by synchrotron radiation X-ray fluorescence imaging.

Two-dimensional distribution of mercury (Hg) in hair samples of rats exposed to methylmercury (MeHg) was analyzed by synchrotron radiation X-ray fluorescence (SR-XRF) imaging. Experiments with endogenous- and exogenous-model for MeHg exposure revealed that the metal level was obviously higher in the hair cortex after the former exposure whereas a dominant site that Hg distributed after the latter exposure was the cuticle. The method also provided us the Hg profile along the hair length with a single hair obtained by the endogenous model. Thus application of SR-XRF analysis to hair sample would facilitate biological monitoring to not only distinct Hg exposure but also determine its dynamics with only the specimen.

Generation of reactive oxygen species during interaction of diesel exhaust particle components with NADPH-cytochrome P450 reductase and involvement of the bioactivation in the DNA damage.

Since the toxicity of diesel exhaust particles (DEP) after intratracheal injection, was suppressed by pretreatment with superoxide dismutase (SOD) modified with polyethylene glycol (Sagai et al. Free Rad. Biol. Med. 14: 37-47; 1993), the possibility that superoxide could be enzymatically and continuously generated from diesel exhaust particles (DEP), was examined. Nicotinamide-adenine dinucleotide phosphate, reduced (NADPH) oxidation was stimulated during interaction of a methanol extract of DEP with the Triton N-101 treated microsomal preparation of mouse lung whereas the cytosolic fraction was less active, suggesting that DEP contains substrates for NADPH-cytochrome P450 reductase (EC 1.6.2.4, P450 reductase) rather than DT-diaphorase. When purified P450 reductase was used as the enzyme source, the turnover value was enhanced approximately 260-fold. Quinones appeared to be served as substrate for P450 reductase because reaction was inhibited by addition of glutathione (GSH) to form those GSH adduct or pretreatment with NaBH4 to reduce those to the hydroxy compounds although a possibility of nitroarenes as the alternative substrates cannot be excluded. A methanol extract of DEP (37.5 micrograms) caused a significant formation of superoxide (3240 nmol/min/mg protein) in the presence of P450 reductase. Electron spin resonance (ESR) experiments revealed that hydroxyl radical was formed as well. The reactive species generated by DEP in the presence of P450 reductase caused DNA scission which was reduced in the presence of superoxide dismutase (SOD), catalase, or hydroxyl radical scavenging agents. Taken together, these results indicate that DEP components, probably quinoid or nitroaromatic structures, that appear to promote DNA damage through the redox cycling based generation of superoxide.

Bioactivation of lapachol responsible for DNA scission by NADPH-cytochrome P450 reductase.

The reduction of the naphthoquinone derivative, lapachol, which is responsible for its bioactivation was examined using microsomal preparations and NADPH-cytochrome P450 reductase (P450 reductase). Phenobarbital (PB) pretreatment resulted in an induction of enzyme activities for cytochrome c reduction (1.54 times) and lapachol reduction (1.20 times) by hepatic microsomal preparation of rats. The specific activity of lapachol reduction by purified P450 reductase showed 56-fold higher than that by untreated liver microsomes. Addition of antibody against P450 reductase (2 mg of IgG/mg of protein) to the microsomal incubation mixture caused an immunoinhibition of cytochrome c (32%) and lapachol (19%) reduction activities, suggesting that P450 reductase catalyzes lapachol reduction. Generation of superoxide anion radical (1321 nmol/mg per min) in approximately equivalent amounts of with NADPH consumption (941 nmol/mg per min) was detected during metabolism of lapachol by P450 reductase. Electron spin resonance (ESR) experiments confirmed generation of superoxide anion radical and hydroxyl radical as these 5,5'-dimethyl-1-pyrroline N-oxide (DMPO) adducts. Incubation of lapachol with P450 reductase caused a cleavage of DNA which was reduced in the presence of Cu,Zn-superoxide dismutase (Cu,Zn-SOD), catalase(1), and hydroxyl radical scavengers such as dimethyl sulfoxide (DMSO) and thiourea. Taken together, these results indicate that lapachol is bioactivated by P450 reductase to reactive species, which promote DNA scission through the redox cycling based generation of superoxide anion radical.

Isozyme selective induction of mouse pulmonary superoxide dismutase by the exposure to mercury vapor.

Alterations in lung superoxide dismutase (SOD) isozymes after exposure of mice to mercury vapor were examined. Inhalation of mercury vapor (10 mg/m(3)) for 1 h by mice resulted in a higher accumulation of mercury in the kidney and lung compared to other organs, at 1 h after exposure. Under these conditions marked enhancement of protein content in bronchoalveolar fluid (BALF), attributed to lung injury, was observed. Exposure to mercury vapor caused a significant increase in the pulmonary Cu,Zn-SOD activity (1.32-fold at 48 h) whereas Mn-SOD activity was suppressed to 82% of the control level, suggesting different sensitivity to the metal inhalation. The selective induction of Cu,Zn-SOD protein (1.79-fold at 48 h) was confirmed by immunoblot analysis with polyclonal antibodies against these isozymes. These observations suggest that the selective induction of Cu,Zn-SOD at the translational level appears to occur as an initial defense against mercury-promoted oxidative stress.

Selective inhibition of the mouse brain Mn-SOD by methylmercury.

Changes in mRNA levels, protein contents and enzyme activities for brain Cu,Zn- and Mn-SOD by methylmercury chloride (MMC) administration, were examined, over a period of 12 days in ICR male mice. After subcutaneous administration of MMC (10 mg/kg) to mice, brain mercury content reached a maximum at 2 days and remained at that level for at least 5 days. MMC exposure resulted in a time-dependent decrease in the Mn-SOD activity: the enzyme activity at 5 days after exposure to MMC was about 60% of control level whereas this exposure was without effect on the Cu,Zn-SOD activity, indicating differential sensitivity of SOD isozymes to the metal. However, levels of mRNA and protein synthesis for Mn-SOD were unaffected by MMC administration. The direct effect of MMC on the both SOD activities were further examined with purified enzyme preparations. After each SOD isozyme (10 U) was incubated with 0.2 mM MMC for 24 h at pH 7.8, the enzyme activities for Cu,Zn- and Mn-SOD were 90% and 37% of control, respectively. Incubations at a ratio of SOD to MMC (1 : 600) for 24 h resulted in a substantial decrease in the enzyme activity of the Mn form; this isozyme-selective inactivation was noted at alkaline pH. A combination of isoelectric focusing-agarose gel electrophoresis (IEF-AGE) and synchrotron radiation X-ray fluorescence (SR-XRF) analysis revealed that Mn-SOD rather than Cu,Zn-SOD underwent modification. Furthermore, a decrease in native form of Mn-SOD protein after MMC exposure was confirmed by gel filtration chromatography. These results indicate that Mn-SOD, but not Cu,Zn-SOD, is susceptible to modification by MMC and the resulting alteration in structure appears to cause a loss of enzyme activities.

An efficient method for purification of cuprozinc superoxide dismutase from bovine erythrocytes.

Cuprozinc superoxide dismutase (Cu,Zn-SOD) was isolated from bovine erythrocytes by pH-controlled ammonium sulfate-methanol extraction (ASME extraction). Adjustment of the pH of a suspension of the lysed red cells in the presence of ammonium sulfate (90% saturation) to pH 5.0, followed by partition with an equal amount of methanol, resulted in isolation of the enzyme with specific activity of greater than 2000 units/mg of protein. Further purification using DEAE-cellulose column chromatography gave a highly purified Cu,Zn-SOD showing a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Using this procedure about 14 mg of pure Cu,Zn-SOD with a specific activity of 4728 units/mg of protein can be recovered from one liter of bovine blood. The enzyme was characterized and the results obtained were in agreement with earlier reports. This procedure appears, therefore, to be a convenient method for isolating the enzyme.