Direct evidence for in vivo hydroxyl radical generation in blood of mice after acute chromium(VI) intake: electron spin resonance spin-trapping investigation.

Although it is assumed from in vitro experiments that the hydroxyl radical (*OH) may be responsible for chromium(VI) toxicity/carcinogenicity, no electron spin resonance (ESR) evidence for the generation of *OH in vivo has been reported. In this study, we have employed an ESR spin-trapping technique with 5,5-dimethylpyrroline-N-oxide (DMPO), a selective *OH trap, to detect *OH in blood. The ESR spectrum of spin adduct observed in the blood of mice given 4.8 mmol Cr(VI)/kg body weight exhibited the 1:2:2:1 intensity pattern of a quartet with a hyperfine coupling constant A(N) = A(H) = 14.81 G and g-value = 2.0067. The concentration of the spin adduct detected in the blood was 7.37 microM. The adduct production was inhibited by the addition of specific *OH scavengers such as sodium benzoate and methional to the blood. The results indicate that the spin adduct is nitroxide produced by the reaction of *OH with DMPO. This is the first report of ESR evidence for the in vivo generation of *OH in mammals by Cr(VI).

In vivo singlet-oxygen generation in blood of chromium(VI)-treated mice: an electron spin resonance spin-trapping study.

Although it is assumed from in vitro experiments that the generation of reactive oxygen species such as the singlet oxygen (1O2), the hydroxyl radical, and the superoxide anion are responsible for chromium(VI) toxicity/carcinogenicity, no electron spin resonance (ESR) evidence for the generation of 1O2 in vivo has been reported. In this study, we have employed an ESR spin-trapping technique with 2,2,6,6-tetramethyl-4-piperidone (TMPD), a specific 1O2 trap, to detect 1O2 in blood. The ESR spectrum of the spin adduct observed in the blood of mice given 4.8 mmol Cr(VI)/kg body weight exhibited the 1:1:1 intensity pattern of three lines with a hyperfine coupling constant A(N) = 16.08 G and a g-value = 2.0066. The concentration of spin adduct detected in the blood was 1.46 microM (0.1% of total Cr concentration). The adduct production was inhibited by the addition of specific 1O2 scavengers such as 1,4-diazabicyclo[2.2.2]octane and sodium azide to the blood. The results indicate that the spin adduct is nitroxide produced by the reaction of 1O2 with TMPD. This is the first report of ESR evidence for the in vivo generation of 1O2 in mammals by Cr(VI).

Cloning of the peroxiredoxin gene family in rats and characterization of the fourth member.

Peroxiredoxin (PRx) exhibits thioredoxin-dependent peroxidase activity and constitutes a family of proteins. Four members of genes from rat tissues were isolated by PCR using degenerated primers based on the sequences which encode a pair of highly conserved Cys-containing domains, and were then cloned to full-length cDNAs. These included two genes which have previously been isolated in rats, PRx I and PRx II, and two rat homologues of PRx III and PRx IV. We showed, for the first time, the simultaneous expression of all four genes in various rat tissues by Northern blotting. Since a discrepancy exists regarding cellular distribution, we further characterized PRx IV by expressing it in COS-1 cells. This clearly demonstrates that PRx IV is a secretory form and functions within the extracellular space.

Immunohistochemical analysis of macrophages, myofibroblasts, and transforming growth factor-beta localization during rat renal interstitial fibrosis following long-term unilateral ureteral obstruction.

Renal interstitial fibrosis was induced in rats by chronic unilateral ureteral obstruction (UUO). To identify the mechanisms behind the fibrosis, macrophage influx, myofibroblast involvement, and the localization of transforming growth factor-beta (TGF-beta, a fibrogenic cytokine) were investigated immunohistochemically in rats euthanatized at 0 (controls), 3, 6, 9, 12, and 15 days after UUO. The number of alpha-smooth muscle actin-positive myofibroblasts began to increase significantly in the medulla from day 3, and the development of medullary fibrosis was confirmed from day 6 by morphometric analysis. From day 9, papillary fibrosis also developed in association with an increased number of myofibroblasts. These myofibroblasts showed a parallel orientation to the mucosal surface of the pelvis. In the medulla and papilla, from day 6 the number of ED1 (primary antibody)-positive macrophages began to increase significantly. There appeared to be a relationship between macrophage influx and myofibroblast involvement. By contrast, in the cortex there was no marked increase in myofibroblasts nor development of fibrotic tissues, regardless of increased number of macrophages from day 6. Immunohistochemically, no staining for TGF-beta was found in infiltrating macrophages or myofibroblasts. However, TGF-beta was localized on some cortical proximal renal tubules both of normal control and obstructed kidneys in the early stages on days 3, 6, and 9, suggesting that the possible origin of TGF-beta may be renal epithelia. However, the staining intensity for TGF-beta on the renal epithelia tended to be weakened in advanced obstructed kidneys on days 12 and 15. The likely contribution of TGF-beta to the advanced stages of UUO-induced renal fibrosis remains to be determined.

Specific detections of the early process of the glycation reaction by fructose and glucose in diabetic rat lens.

The glycation reaction by fructose, as well as that by glucose, in control and diabetic rat lens was analyzed by using antibodies which specifically recognize adducts of lysine with fructose and with glucose. Levels of fructose adducts in diabetic rat lens were 2.5 times that of the control, and correlated with sorbitol levels. This was mainly due to enhanced glycation of beta- and gamma-crystallins by fructose under diabetic conditions. These data suggest that glycation by fructose may also play a role in cataract formation under conditions of diabetes and aging.

Selective induction of heparin-binding epidermal growth factor-like growth factor by methylglyoxal and 3-deoxyglucosone in rat aortic smooth muscle cells. The involvement of reactive oxygen species formation and a possible implication for atherogenesis in diabetes.

Methylglyoxal (MG) and 3-deoxyglucosone (3-DG), reactive dicarbonyl metabolites in the glyoxalase system and glycation reaction, respectively, selectively induced heparin-binding epidermal growth factor (HB-EGF)-like growth factor mRNA in a dose- and time-dependent manner in rat aortic smooth muscle cells (RASMC). A nuclear run-on assay revealed that the dicarbonyl may regulate expression of HB-EGF at the transcription level. The dicarbonyl also increased the secretion of HB-EGF from RASMC. However, platelet-derived growth factor, another known growth factor of smooth muscle cells (SMC), was not induced by both dicarbonyls. The dicarbonyl augmented intracellular peroxides prior to the induction of HB-EGF mRNA as judged by flow cytometric analysis using 2',7'-dichlorofluorescin diacetate. N-Acetyl-L-cysteine and aminoguanidine suppressed both dicarbonyl-increased HB-EGF mRNA and intracellular peroxide levels in RASMC. DL-Buthionine-(S, R)-sulfoximine increased the levels of 3-DG-induced HB-EGF mRNA. Furthermore, hydrogen peroxide alone also induced HB-EGF mRNA in RASMC. These results indicate that MG and 3-DG induce HB-EGF by increasing the intracellular peroxide levels. In addition, the pretreatment with 12-O-tetra-decanoylphorbol-13-acetate failed to alter dicarbonyl-induced HB-EGF mRNA expression in RASMC, suggesting that the signal transducing mechanism is not mediated by protein kinase C. Since HB-EGF is known as a potent mitogen for smooth muscle cells and is abundant in atherosclerotic plaques, the induction of HB-EGF by MG and 3-DG, as well as the concomitant increment of intracellular peroxides, may trigger atherogenesis during diabetes.

Roles of purine nucleotides and adenosine in enhancing NOS II gene expression in interleukin-1 beta-stimulated rat vascular smooth muscle cells.

The production of nitric oxide (NO) by vascular smooth muscle cells (VSMC) is stimulated by interleukin-1 beta (IL-1 beta). This is enhanced in a dose-dependent manner by ADP, although it alone failed to induce nitrite accumulation. Purine nucleotides and their nonhydrolizable analogues as well as adenosine also exhibit variable enhancing effects. This enhanced nitrite formation was due to induction of the NO synthase (NOS II) gene as judged by Northern hybridization using an NOS II specific probe and by Ca2+ independency of the NOS II activity. 8-(p-Sulfophenyl)-theophylline, a blocker of adenosine receptors, suppressed the enhanced NO production by adenosine and ADP to the level of that with IL-1 beta alone. These data indicate that activation of the adenosine receptor on VSMC may enhance production of NOS II by modulating a signal transducing pathway of IL-1 beta. Although cAMP is a candidate as the second messenger, it was not significantly elevated by either ADP or adenosine treatment in IL-1 beta-stimulated cells. This mechanism might be stimulated under conditions with release of various purine and their derivatives.

Quinazoline derivatives suppress nitric oxide production by macrophages through inhibition of NOS II gene expression.

We have found three novel quinazolidine derivatives which inhibit the formation of nitrite dose-dependently in a murine macrophage cell line, RAW264.7. The decreased nitrite formation was due not to the inhibition of nitric oxide synthase activity but to suppression of NOS II mRNA and protein expression. In rat vascular smooth muscle cells (VSMC), however, these compounds rather enhanced NOS II mRNA. These compounds also prevented LPS-stimulated heme oxygenase-1 (HO-1) and cyclooxygenase-2 (COX-2) gene expression in RAW264.7 cells, but again not in VSMC. The three quinazolidine derivatives specifically inhibit gene expression of NOS II, HO-1 and COX-2 only in macrophage cells, indicating that they are selective inhibitors of inducible gene expression in macrophages.

Induction of apoptotic cell death by methylglyoxal and 3-deoxyglucosone in macrophage-derived cell lines.

Production of 2-oxoaldehyde compounds increase during hyperglycemic conditions and is cytotoxic to susceptible cells. We have investigated the effects of methylglyoxal and 3-deoxyglucosone at physiological concentrations on monocytic leukemia U937 cells and other cell lines. Both ladder formation of DNA and nuclear fragmentation were observed in the cells treated with these agents, indicating that apoptotic cell death was induced. The fluorescent intensity of an oxidation sensitive dye (2',7'-dichlorofluorescin) was increased in U937 cells but not in other cells in which apoptosis was not induced. The levels of intracellular glutathione, however, were only slightly changed. Apoptosis and intracellular oxidant levels were enhanced by buthionine sulfoximine, an inhibitor of glutathione biosynthesis, and partially blocked by N-acetylcysteine, an antioxidant. Thus, it is conceivable that elevation of intracellular oxidant stress is a cause of the apoptosis induced by cytotoxic 2-oxoaldehyde compounds.

Oxidative stress caused by glycation of Cu,Zn-superoxide dismutase and its effects on intracellular components.

It is now evident that the redox state of the cell is a pivotal determinant of the fate of cells. Extensive production of reactive oxygen species (ROI) causes necrotic cell death. Even transient or localized production of ROI may mediate a signal for apoptotic cell death, whereas small amounts of ROI function as an intracellular messenger of some growth stimulants. Accumulating evidence supports the concept that decreases in Cu,Zn-superoxide dismutase (SOD) activity causes apoptotic cell death in neuronal cells. Our data using mutant Cu,Zn-SOD related to familial amyotrophic lateral sclerosis (FALS) suggest that glycation itself and ROI produced from the glycated proteins are involved in many diseases, including diabetic complications. Glycation of important cellular components, including lipid, DNA and proteins, induces dysfunction of these components. Mutant proteins in patients with various hereditary diseases would be destabilized by the glycation reaction, as shown in the case of mutant Cu,Zn-SODs, thereby hyperglycaemic conditions would trigger the onset of some hereditary diseases such as FALS and Alzheimer's disease. Glycation, particularly of antioxidative enzymes, would enhance production of ROI, resulting in oxidative damage to the cells.