Distribution and speciation of arsenic after intravenous administration of monomethylmonothioarsonic acid in rats.

Quite a few new thioarsenicals have recently been found in urine of arsenic-exposed humans and animals, and some of them have been shown to be highly toxic to cells. However, little is known about their toxic effects and metabolism in the body. In order to elucidate the toxic mechanism of thioarsenicals, we further focused on the distribution and metabolism of monomethylmonothioarsonic acid (MMMTA(V)) in rats. MMMTA(V) was synthesized chemically and injected intravenously into rats at the dose of 0.5mg As/kg, followed by speciation analysis of selected organs and body fluids at 10 min and 12h after the injection. MMMTA(V) was excreted into urine in its intact form, and approximately 35% of the dose was recovered in urine at 12h after the injection, suggesting that MMMTA(V) was taken up more effectively by organs/tissues than non-thiolated, monomethylarsonous acid (MMA(V)) previously studied. On the other hand, the liver and kidneys contained arsenic that was in a protein-binding form with free forms of DMA(V) or DMDTA(V) at 10 min, and disappeared at 12h after the injection. Moreover, these bound arsenic species in kidneys were converted back to MMA(V) after oxidation with H(2)O(2), suggesting that the arsenic bound to proteins had been reduced within the body and was in a trivalent oxidation state. In red blood cells (RBCs), most of the arsenic was in the form of DMA(III) bound to hemoglobin (Hb), and approximately 40% of the dose was recovered in RBCs at 12h after injection. These results indicate that arsenic accumulated preferentially in RBCs after being transformed to DMA(III). In addition, we have also discussed the effect of MMMTA(V) on viability of human bladder cancer T24 cells in comparison with MMA(V). Consequently, MMMTA(V) was assumed to be a more toxic arsenic metabolite than non-thiolated MMA(V).

Evidence for toxicity differences between inorganic arsenite and thioarsenicals in human bladder cancer cells.

Arsenic toxicity is dependent on its chemical species. In humans, the bladder is one of the primary target organs for arsenic-induced carcinogenicity. However, little is known about the mechanisms underlying arsenic-induced carcinogenicity, and what arsenic species are responsible for this carcinogenicity. The present study aimed at comparing the toxic effect of DMMTA(V) with that of inorganic arsenite (iAs(III)) on cell viability, uptake efficiency and production of reactive oxygen species (ROS) toward human bladder cancer EJ-1 cells. The results were compared with those of a previous study using human epidermoid carcinoma A431 cells. Although iAs(III) was known to be toxic to most cells, here we show that iAs(III) (LC(50)=112 microM) was much less cytotoxic than DMMTA(V) (LC(50)=16.7 microM) in human bladder EJ-1 cells. Interestingly, pentavalent sulfur-containing DMMTA(V) generated a high level of intracellular ROS in EJ-1 cells. However, this was not observed in the cells exposed to trivalent inorganic iAs(III) at their respective LC(50) dose. Furthermore, the presence of N-acetyl-cysteine completely inhibited the cytotoxicity of DMMTA(V) but not iAs(III), suggesting that production of ROS was the main cause of cell death from exposure to DMMTA(V), but not iAs(III). Because the cellular uptake of iAs(III) is mediated by aquaporin proteins, and because the resistance of cells to arsenite can be influenced by lower arsenic uptake due to lower expression of aquaporin proteins (AQP 3, 7 and 9), the expression of several members of the aquaporin family was also examined. In human bladder EJ-1 cells, mRNA/proteins of AQP3, 7 and 9 were not detected by reverse transcription polymerase chain reaction (RT-PCR)/western blotting. In A431 cells, only mRNA and protein of AQP3 were detected. The large difference in toxicity between the two cell lines could be related to their differences in uptake of arsenic species.

Identification of the major arsenic-binding protein in rat plasma as the ternary dimethylarsinous-hemoglobin-haptoglobin complex.

Chronic exposure to arsenic causes a wide range of diseases such as hyperkeratosis, cardiovascular diseases, and skin, lung, and bladder cancers, and millions of people are chronically exposed to arsenic worldwide. However, little is known about the mechanisms underlying these toxic actions. The metabolism of arsenic is essential for understanding the toxic actions. Here, we identified the major arsenic-binding protein (As-BP) in the plasma of rats after oral administration of arsenite by the use of two different HPLC columns, gel filtration and anion exchange ones, coupled with an inductively coupled argon plasma mass spectrometer (ICP MS). The molecular mass of the As-BP was estimated to be 90 kDa based on results using the former column, and arsenic bound to this protein only in the form of dimethylarsinous acid (DMA (III)) in the plasma in vivo. In addition, the purified As-BP was shown to consist of two different proteins, haptoglobin (Hp) of 37 kDa (three bands) and the hemoglobin (Hb) alpha chain of 14 kDa (single band), using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS), respectively, suggesting that the As-BP was the ternary DMA (III)-Hb-Hp complex. To confirm the present observations, an arsenic-binding assay was carried out in vitro . Although DMA (III) bound directly to fresh rat plasma proteins, they were different from that identified in vivo. However, when a DMA (III)-exposed rat RBC lysate (DMA (III) binds to Hb in rat RBCs) was added to control rat plasma, a new arsenic peak increased at the expense of the arsenic-Hb one. Furthermore, this new arsenic peak was consistent with the As-BP identified in the plasma in vivo, suggesting that arsenic bound to Hb further binds to haptoglobin (Hp), forming the ternary As-Hb-Hp complex.

Theoretical calculations and reaction analysis on the interaction of pentavalent thioarsenicals with biorelevant thiol compounds.

To obtain a rational understanding of the extraordinary interaction of pentavalent thioarsenicals with biorelevant thiol compounds, we carried out ab initio calculations on related arsenic compounds and discussed the correlation between the distribution of observed arsenic species in actual reaction systems and the corresponding calculated reaction enthalpies. Previously, it was considered that pentavalent arsenicals do not form thiol conjugates. However, the dimethylmonothioarsinic acid-glutathione conjugate (DMMTAV-GSH) was recently reported as the first stable conjugate of a pentavalent arsenical with a thiol compound. We carried out detailed analysis of the DMMTAV-GSH formation reaction and demonstrated that this conjugate could be formed nonenzymatically under weakly acidic conditions. On the basis of the ab initio calculations, this conjugation was an exothermic reaction (delta H = -4.85 kcal/mol) and gave the minimum energy point during the reaction sequence of DMMTAV with a thiol compound. However, in the case of dimethylarsinic acid (DMAV), a corresponding oxo acid to DMMTAV, conjugation with a thiol compound is an endothermic reaction (delta H = +0.06 kcal/mol). The minimum energy point of the reaction sequence of DMAV with a thiol compound was the formation of a trivalent dimethylarsinous acid (DMAIII)-GSH conjugate. Because the formation of arsenic-sulfur bonds is one of the major mechanisms for arsenic toxicity, these energetic results could account for the extraordinary behaviors and toxicities of thioarsenicals in vivo and in vitro in comparison with those of the corresponding oxo acids.

Formation of dimethylthioarsenicals in red blood cells.

The bladder and skin are the primary targets for arsenic-induced carcinogenicity in mammals. Thioarsenicals dimethylmonothioarsinic (DMMTA(V)) and dimethyldithioarsinic (DMDTA(V)) acids are common urinary metabolites, the former being much more toxic than non-thiolated dimethylarsinic acid (DMA(V)) and comparable to dimethylarsinous acid (DMAIII) in epidermoid cells, suggesting that the metabolic production of thioarsenicals may be a risk factor for the development of cancer in these organs. To reveal their production sites (tissues/body fluids), we examined the uptake and transformation of the four dimethylated arsenicals by incubation with rat and human red blood cells (RBCs). Although DMA(V) and DMDTA(V) were not taken up by either type of RBCs, DMAIII and DMMTA(V) were taken up by both (more efficiently by rat ones), though DMMTA(V) was taken up slowly, and then the arsenic transformed into DMDTA(V) was excreted from both types of animal RBCs. On the other hand, although DMA(III) taken up rapidly by rat RBCs was retained in the RBCs, that taken up by human RBCs was immediately transformed into DMMTA(V) and then excreted into the incubation medium without being retained in the RBCs. In a separate experiment, arsenic remaining in primary rat hepatocytes after incubation with 1.5 microM DMAIII was recovered from the incubation medium in the forms of DMA(V) and DMMTA(V) in the presence of human RBCs, but not in the presence of rat RBCs (in which the arsenic was bound to hemoglobin). Thus, DMMTA(V) was detected in the medium only in the presence of human RBCs and increased with incubation time. It was proposed that arsenic is excreted from hepatocytes into the bloodstream in the form of DMAIII and then taken up by RBCs in humans, where it is transformed into DMMTA(V) and then excreted again into the bloodstream.

Methylation and demethylation of intermediates selenide and methylselenol in the metabolism of selenium.

All nutritional selenium sources are transformed into the assumed common intermediate selenide for the syntheses of selenoproteins for utilization and/or of selenosugar for excretion. Methylselenol [monomethylselenide, MMSe] is the assumed intermediate leading to other methylated metabolites, dimethylselenide (DMSe) and trimethylselenonium (TMSe) for excretion, and also to the intermediate selenide from methylselenocysteine and methylseleninic acid (MSA). Here, related methylation and demethylation reactions were studied in vitro by providing chemically reactive starting substrates (76Se-selenide, 77Se-MMSe and 82Se-DMSe) which were prepared in situ by the reduction of the corresponding labeled proximate precursors (76Se-selenite, 77Se-MSA and 82Se-dimethylselenoxide (DMSeO), respectively) with glutathione, the three substrates being incubated simultaneously in rat organ supernatants and homogenates. The resulting chemically labile reaction products were detected simultaneously by speciation analysis with HPLC-ICP-MS after converting the products and un-reacted substrates to the corresponding oxidized derivatives (selenite, MSA and DMSeO). The time-related changes in selenium isotope profiles showed that demethylation of MMSe to selenide was efficient but that of DMSe to MMSe was negligible, whereas methylation of selenide to MMSe, and MMSe to DMSe were efficient, and that of DMSe to TMSe occurred less efficiently. The present methylation and demethylation reactions on equilibrium between selenide, MMSe and DMSe without producing selenosugar and selenoproteins indicated that DMSe rather than TMSe is produced as the end product, suggesting that DMSe is to be excreted more abundantly than TMSe. Organ-dependent differences in the methylation and demethylation reactions were characterized for the liver, kidney and lung.

Preferential organ distribution of methylselenol source Se-methylselenocysteine relative to methylseleninic acid.

It has been proposed that Se-methylselenocysteine (MeSeCys) and methylseleninic acid (MSA(IV)) are efficiently transformed through the beta-lyase and reduction reactions, respectively, into methylselenol, the assumed biologically active selenometabolite responsive for the anti-carcinogenicity and anti-oxidant actions of selenium. The bioavailability and distribution of the two selenium sources in major organs/tissues were compared under exactly identical conditions. Namely, labeled selenium sources (76)Se-MeSeCys and (77)Se-MSA(IV), at a single oral dose of 10 microg Se/kg body weight each, were administered simultaneously to rats that had been depleted of natural abundance selenium with a single isotope (78)Se. The same dose of (82)Se-selenite was also administered as a reference selenium source. The distributions of the three labeled selenium isotopes were determined 3 h after the administration in 13 organs/tissues/blood. MeSeCys was taken up more efficiently by most organs, especially the pancreas and duodenum, than MSA(IV) and selenite, the latter two sources being taken up similarly to each other except for in the kidney, liver, and spleen, where the three labeled isotopes were detected at comparable concentrations. The labeled selenium in the liver supernatant was speciated by HPLC inductively coupled argon plasma-mass spectrometry (ICP-MS), and it was suggested that MeSeCys was delivered in its intact form to organs, and then transformed into methylselenol. In addition to the known properties of that MeSeCys is chemically more stable than MSA(IV) and is a naturally occurring edible product, and that MeSeCys produces methylselenol much more efficiently than a homologous selenoamino acid selenomethionine, the present study revealed that MeSeCys is more efficiently distributed than MSA(IV) in its intact form, and then produces methylselenol, suggesting that MeSeCys is the best methylselenol source in most organs/tissues.

Toxicity of dimethylmonothioarsinic acid toward human epidermoid carcinoma A431 cells.

Chronic ingestion of arsenic-contaminated drinking water induces skin lesions and urinary bladder cancer in humans. It is now recognized that thioarsenicals such as dimethylmonothioarsinic acid (DMMTA (V)) are commonly excreted in the urine of humans and animals and that the production of DMMTA (V) may be a risk factor for the development of the diseases caused by arsenic. The toxicity of DMMTA (V) was compared with that of related nonthiolated arsenicals with respect to cell viability, uptake ability, generation of reactive oxygen species (ROS), and cell cycle progression of human epidermoid carcinoma A431 cells, arsenate (iAs (V)), arsenite (iAs (III)), dimethylarsinic acid (DMA (V)), and dimethylarsinous acid (DMA (III)) being used as reference nonthiolated arsenicals. DMMTA (V) (LC 50 = 10.7 microM) was shown to be much more cytotoxic than iAs (V) (LC 50 = 571 microM) and DMA (V) (LC 50 = 843 microM), and its potency was shown to be close to that of trivalent arsenicals iAs (III) (LC 50 = 5.49 microM) and DMA (III) (LC 50 = 2.16 microM). The greater cytotoxicity of DMMTA (V) was associated with greater cellular uptake and distribution, and the level of intracellular ROS remarkably increased in A431 cells upon exposure to DMMTA (V) compared to that after exposure to other trivalent arsenicals at the respective LC 50. Exposure of DMMTA (V) to cells for 24 h induced cell cycle perturbation. Namely, the percentage of cells residing in S and G2/M phases increased from 10.2 and 15.6% to 46.5 and 20.8%, respectively. These results suggest that although DMMTA (V) is a pentavalent arsenical, it is taken up efficiently by cells and causes various levels of toxicity, in a manner different from that of nonthiolated pentavalent arsenicals, demonstrating that DMMTA (V) is one of the most toxic arsenic metabolites. The high cytotoxicity of DMMTA (V) was explained and/or proposed by (1) efficient uptake by cells followed by (2) its transformation to DMA (V), (3) producing ROS in the redox equilibrium between DMA (V) and DMA (III) in the presence of glutathione.

Metabolism of 76Se-methylselenocysteine compared with that of 77Se-selenomethionine and 82Se-selenite.

Se-Methylated selenoamino acids, Se-methylselenocysteine (MeSeCys) and selenomethionine (SeMet), are chemically inert storage forms of selenium in selenium-accumulators, and a nutritional and supplemental source. The metabolic pathway for MeSeCys was precisely traced by referring to those for SeMet and selenite by applying a new tracer method involving multiple homo-elemental stable isotopes. Male Wistar rats were depleted of endogenous natural abundance selenium with a single (80)Se-enriched isotope, and then (76)Se-MeSeCys, (77)Se-SeMet and (82)Se-selenite were orally administered simultaneously at 25 microg Se/kg body weight each. Organs and body fluids were obtained at 3, 6, 9 and 12 h, and 1 and 2 days later, and subjected to speciation analysis. The main characteristics of the metabolism were as follows; MeSeCys was incorporated into selenoprotein P slightly more than or at a comparable level to that of SeMet but less than that of selenite. MeSeCys and SeMet but not selenite was taken up by organs in their intact forms. MeSeCys and SeMet were delivered specifically to the pancreas and present in a form bound to an identical or similar protein. Trimethylselenonium (TMSe) was only produced from MeSeCys, i.e., not from SeMet or selenite, in the kidneys. Both selenosugars A and B of MeSeCys, SeMet and selenite origin were detected in the liver but only selenosugar B in the kidneys. These results suggest that MeSeCys can be a similar or better selenium source than SeMet, and supplies methylselenol much more efficiently in organs than SeMet and selenite. TMSe was produced much efficiently from MeSeCys than from SeMet and selenite, suggesting a role of methylselenol through the beta-lyase reaction in the metabolism of Se-methylated selenoamino acids.

Protective role of metallothionein against copper depletion.

Copper (Cu) is one of the essential metals and its homeostasis is strictly regulated. Metallothionein (MT) is induced by excess Cu to mask the Cu toxicity. Although the role of MT in Cu toxicity has been explained in terms of Cu sequestration, its role under Cu-deficient conditions is not known. This study was carried out to determine the role of MT in Cu depletion by a Cu(I)-specific chelator, bathocuproine sulfonate (BCS), in cultured cells established from MT-knockout mouse and its wild type. Viability was decreased more severely in MT-null cells than in wild-type cells by BCS treatment. The expression levels of both MT isoforms were increased by BCS treatment in wild-type cells. Thus, MT was shown to be induced under Cu-deficient conditions to maintain the activities of intracellular cuproenzymes such as cytochrome c oxidase and Cu,zinc-superoxide dismutase.

Reaction of para-hydroxybenzoic acid esters with singlet oxygen in the presence of glutathione produces glutathione conjugates of hydroquinone, potent inducers of oxidative stress.

The determination and toxicological characterization of products of the reaction between p-hydroxybenzoic acid esters (parabens) and singlet oxygen ((1)O(2)) are very important because of the frequent use of parabens in cosmetics and possible generation of (1)O(2) in the skin. We observed (1)O(2)-dependent production of mono-, di-, and tri-substituted glutathione (GSH) conjugates of hydroquinone (HQ) during visible light-irradiation of a mixture of methyl or ethyl paraben and GSH in the presence of rose bengal (RB). 1,4-Benzoquinone (BQ) and HQ were produced during the irradiation in the absence of GSH. While a mixture of BQ and GSH produced only mono-substituted conjugate, irradiation of the mixture with RB produced mono-, di-, and tri-substituted conjugates. These observations indicate that (1)O(2) is involved both in the production of BQ and HQ from parabens and in the formation of multi-substituted GSH conjugates from mono-substituted conjugate. Tri-substituted conjugate generated larger amounts of hydrogen peroxide in an aqueous solution than mono-substituted conjugates or HQ did. Detection of semiquinone radical suggests that the autoxidation of conjugates is related to the generation of hydrogen peroxide. The results obtained in this study indicate that parabens may induce oxidative stress in the skin after conversion to GSH conjugates of HQ by reacting with (1)O(2) and GSH.

Liquid chromatography-mass spectrometry (LC-MS): a powerful combination for selenium speciation in garlic (Allium sativum).

Liquid chromatography (LC) hyphenated with both elemental and molecular mass spectrometry has been used for Se speciation in Se-enriched garlic. Different species were separated by ion-pair liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS) after hot-water extraction. They were identified by on-line reversed-phase liquid chromatography-electrospray ionization tandem mass spectrometry (RPLC-ESI-MS-MS). Se-methionine and Se-methylselenocysteine were determined by monitoring their product ions. Another compound, gamma-glutamyl-Se-methylselenocysteine, shown to be the most abundant form of Se in the garlic, was determined without any additional sample pre-treatment after extraction and without the need for a synthesized standard. Product ions for this dipeptide were detected by LC-ESI-MS-MS for three isotopes of Se-78 Se, 80Se: and 82Se. The method was extended to the species extracted during in-vitro gastrointestinal digestion. Because both Se-methylselenocysteine and gamma-glutamyl-Se-methylselenocysteine have anticarcinogenic properties, their extractability and stability during human digestion are very important. Garlic was also treated with saliva, to enable detection and analysis of species extracted during mastication. Detailed information on the extractability of selenium species by both simulated gastric and intestinal fluid are given, and variation of the distribution of Se among the different species with time is discussed. Although the main species in garlic is the dipeptide gamma-glutamyl-Se-methylselenocysteine, Se-methylselenocysteine is the main compound present in the extracts after treatment with gastrointestinal fluids. Two more, so far unknown compounds were observed in the chromatogram. The extracted species and their transformations were analysed by combining LC-ICP-MS and LC-ESI-MS-MS. In both the simulated gastric and intestinal digests, Se-methionine, Se-methylselenocysteine, and gamma-glutamyl-Se-methylselenocysteine could be determined by LC-ESI-MS-MS by measuring their typical product ions.

Simultaneous speciation of selenium and sulfur species in selenized odorless garlic (Allium sativum L. Shiro) and shallot (Allium ascalonicum) by HPLC-inductively coupled plasma-(octopole reaction system)-mass spectrometry and electrospray ionization-tandem mass spectrometry.

The simultaneous speciation of selenium and sulfur in selenized odorless garlic (Allium sativum L. Shiro) and a weakly odorous Allium plant, shallot (Allium ascalonicum), was performed by means of a hyphenated technique, a HPLC coupled with an inductively coupled plasma-mass spectrometry (HPLC-ICP-MS) equipped with an octopole reaction system (ORS). The aqueous extracts of them contained the common seleno compound that was identified as gamma-glutamylmethylselenocysteine by an electrospray ionization-tandem mass spectrometry (ESI-MS/MS). Normal garlic contains alliin as the major sulfur-containing compound, which is the biological precursor of the garlic odorant, allicin. Alliin, however, was not detected in the extracts of the selenized odorless garlic. At least, four unidentified sulfur-containing compounds were detected in odorless garlic and shallot. Moreover, these Allium plants showed chemopreventive effects against human leukemia cells.

Hydroxyl radical generation via photoreduction of a simple pyridine N-oxide by an NADH analogue.

Photoreduction of pyridine N-oxide, which has a key structure of antitumor agents for hypoxic solid tumors, by 1-benzyl-1,4-dihydronicotinamide in deaerated aprotic media resulted in generation of hydroxyl radical, leading to the oxidation of salicylic acid to 2,3- and 2,5-dihydroxybenzoic acids, and catechol.

Early pathophysiological features in canine renal papillary necrosis induced by nefiracetam.

To ascertain the early pathophysiological features in canine renal papillary necrosis (RPN) caused by the neurotransmission enhancer nefiracetam, male beagle dogs were orally administered nefiracetam at 300 mg/kg/day for 4 to 7 weeks in comparison with ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), at 50 mg/kg/day for 5 weeks. During the dosing period, the animals were periodically subjected to laboratory tests, light-microscopic, immunohistochemical, and electron-microscopic examinations and/or cyclooxygenase (COX)-2 mRNA analysis. In laboratory tests, a decrease in urinary osmotic pressure and increases in urine volume and urinary lactate dehydrogenase (LDH) level were early biomarkers for detecting RPN. Light-microscopically, nefiracetam revealed epithelial swelling and degeneration in the papillary ducts in week 7, while ibuprofen displayed degeneration and necrosis in the papillary interstitium in week 5. In immunohistochemical staining with COX-2 antibody, nefiracetam elicited a positive reaction within interstitial cells around the affected epithelial cells in the papillary ducts (upper papilla) in week 7, and ibuprofen positively reacted within interstitial cells adjacent to the degenerative and/or necrotic lesions in week 5. Ultrastructurally, nefiracetam exhibited reductions of intracellular interdigitation and infoldings of epithelial cells in the papillary ducts, whereas ibuprofen showed no changes in the identical portions. Thus, the early morphological change in the papilla brought about by nefiracetam was quite different from that elicited by ibuprofen. By the renal papillary COX-2 mRNA expression analysis, nefiracetam exceedingly decreased its expression in week 4, but markedly increased it in week 7, suggesting an induction of COX-2 mRNA by renal papillary lesions. These results demonstrate that the epithelial cell in the papillary ducts is the primary target site for the onset of RPN evoked by nefiracetam.

Investigation on urinary proteins and renal mRNA expression in canine renal papillary necrosis induced by nefiracetam.

The occurrence of renal papillary necrosis (RPN), seen only in dogs after repeated oral administration of nefiracetam, a neurotransmission enhancer, at a relatively high dose, is because of inhibition of renal prostaglandin synthesis by the nefiracetam metabolite M-18. In this study, analyses of urinary proteins and renal mRNA expression were performed to investigate the possible existence of a specific protein expressing the characteristics of RPN evoked by nefiracetam. In the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of urinary proteins from male dogs given nefiracetam at 300 mg kg(-1) day(-1) over weeks 5-11, a protein of approximately 40 kDa, which was not seen in control urine, and protein of approximately 30 kDa emerged as distinct bands. Subsequently, clusterin precursor was identified in the former band and tissue kallikrein precursor in the latter by LC-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS). By quantitative real-time RT-PCR analysis with renal morphological aspects, individual findings showed that renal clusterin mRNA was increased in dogs with severe renal injury, and renal tissue kallikrein also increased, presumably related to hemodynamics. These results demonstrate that changes in renal clusterin mRNA may reflect the progression or severity of RPN, whereas upregulation of tissue kallikrein mRNA may subsequently play a compensating role in the prevention of RPN.

Nuclear trafficking of metallothionein requires oxidation of a cytosolic partner.

The present study revealed the mechanism underlying the nuclear trafficking of metallothionein (MT). Nuclear localization of MT in digitonin-permeabilized BALB 3T3 cells was enhanced in the presence of a cytosolic factor added as a rat red blood cell lysate by oxidation with H2O2 in a dose-dependent manner, but inhibited with excess glutathione. A cytosolic partner was assumed to bind MT and retain it in the cytoplasm, and its oxidation can mobilize MT to the nuclei on cellular oxidation. Pre-treatment of nuclei with H2O2 did not enhance the localization, and MT that had been localized in the nuclei was washed out, indicating that MT is in the nuclei as a result of a higher rate of uptake by the nuclei than the rate of diffusion from the nuclei. Nuclear localization of lysozyme and nuclear localization signal (NLS)-bearing allophycocyanin were not enhanced by the oxidation in the presence of cytosolic factor, suggesting that the nuclear traffic occurring on oxidation is specific to MT. Moreover, when cells were arrested the cell cycle at the S phase, MT was localized in the nuclei in response to coincidental generation of a feeble reactive oxygen species (ROS). These observations suggest that MT comes localized in the nuclei on the sensing of intracellular oxidation, whereby a cytosolic partner specific to MT comes oxidized as a cargo system, MT being localized as a result of enhanced uptake in the nuclei and re-localized in the cytoplasm diffusely. Nuclear MT was proposed to protect the nuclei from the oxidation occurring with progression of the cell cycle.

Hydroxyl radical generation caused by the reaction of singlet oxygen with a spin trap, DMPO, increases significantly in the presence of biological reductants.

Photosensitizers newly developed for photodynamic therapy of cancer need to be assessed using accurate methods of measuring reactive oxygen species (ROS). Little is known about the characteristics of the reaction of singlet oxygen (1O2) with spin traps, although this knowledge is necessary in electron spin resonance (ESR)/spin trapping. In the present study, we examined the effect of various reductants usually present in biological samples on the reaction of 1O2 with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). The ESR signal of the hydroxyl radical (*OH) adduct of DMPO (DMPO-OH) resulting from 1O2-dependent generation of *OH strengthened remarkably in the presence of reduced glutathione (GSH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), ascorbic acid, NADPH, etc. A similar increase was observed in the photosensitization of uroporphyrin (UP), rose bengal (RB) or methylene blue (MB). Use of 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) as a spin trap significantly lessened the production of its *OH adduct (DEPMPO-OH) in the presence of the reductants. The addition of DMPO to the DEPMPO-spin trapping system remarkably increased the signal intensity of DEPMPO-OH. DMPO-mediated generation of *OH was also confirmed utilizing the hydroxylation of salicylic acid (SA). These results suggest that biological reductants enhance the ESR signal of DMPO-OH produced by DMPO-mediated generation of *OH from 1O2, and that spin trap-mediated *OH generation hardly occurs with DEPMPO.

Synchronized generation of reactive oxygen species with the cell cycle.

A possible appearance of reactive oxygen species (ROS) with the normal cell cycle was studied to find how ROS are generated in cells in relation to the cell cycle. The production of ROS in relation to the cell cycle was examined by determining the changes in intracellular ROS concentrations at different phases of the cell cycle by culturing BALB 3T3 cells in the presence and absence of aphidicolin. The amounts of intracellular ROS and the cell population at specific phases (S and G2/M) were determined as the fluorescence of dichlorodihydrofluorescein and propidium iodide taken up simultaneously by the cells, respectively, by flow cytometry. Although intracellular ROS remained at the control levels when the cell growth was arrested with aphidicolin at the G1 phase, they increased when the arrest was released to result in the increase of the cell population at the S phase. Furthermore, ROS was shown to disturb/stop the cell cycle by means of the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. The cell cycle was regulated through oxidative stress by exposure to hydrogen peroxide and glutathione ethyl ester. The cell cycle was prevented more sensitively in metallothionein-null cells than in the wild type cells. Based on the present observations, we proposed for the first time that ROS are generated synchronously with the normal cell cycle, and that they have to be controlled at certain level for normal progress of the cell cycle.

Heavy metal tolerance of transgenic tobacco plants over-expressing cysteine synthase.

Cysteine synthase [O-acetyl-L-serine(thiol)lyase] catalyzes the final step for L-cysteine biosynthesis in plants. The tolerance of transgenic tobacco plants over-expressing cysteine synthase cDNA in cytosol (3F), chloroplasts (4F) and in both organelles (F1) was investigated towards heavy metals such as Cd, Se, Ni, Pb and Cu. The transgenic plants were significantly more tolerant than wild-type plants in agar medium containing Cd, Se and Ni. The F1 transgenic plants had a higher resistance than other transgenic lines towards these metals and could enhance accumulation of Cd in shoot. These results suggest that the transgenic plants over-expressing cysteine synthase both in cytosol and chloroplasts can be applicable to phyto-remediation of Cd from contaminated soils.