Nobuhiro Shimojo

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.

Decreased serum concentrations of nitric oxide metabolites among Chinese in an endemic area of chronic arsenic poisoning in inner Mongolia

Prolonged exposure to arsenic results in peripheral and cardiovascular manifestations, as does impaired production of endothelial nitric oxide (NO). In vitro studies have indicated that endothelial cells undergo damage by arsenic. However, no information has been available on the relationship between NO synthesis and chronic arsenic poisoning in humans. The present study was designed to reveal this question. The subjects were 33 habitants who continued to drink well water containing high concentrations of inorganic arsenic (mean value = 0.41 microg/ml) for about 18 years in Inner Mongolia, China, and 10 other people who lived in this area but exposed to minimal concentrations of arsenic (mean value = 0.02 microg/ml) were employed as controls. Mean blood concentration of total arsenic was six times higher in exposed subjects than controls; 42.1 vs. 7.3 ng/ml, p <.001. Mean serum concentration of nitrite/nitrate, stable metabolites of endogenous NO, was lower in arsenic-exposed subjects than in controls: 24.7 vs. 51.6 microM, p<.001. In total samples, an inverse correlation with serum nitrite/nitrate levels was strong for blood inorganic arsenic (r = -0.52, p <.001) and less strong for its metabolites, monomethyl arsenic (r = -0.45, p<.005) and dimethyl arsenic (r = -0.37, p<.05). Furthermore, serum nitrite/nitrate concentration was significantly correlated with nonprotein sulfhydryl level in whole blood (r = 0.58, p<.001). In an in vitro study, we demonstrated that inorganic arsenite or arsenate suppresses the activity of endothelial NO synthase in human umbilical vein endothelial cells. These results suggest that long-term exposure to arsenic by drinking well water possibly reduces NO production in endothelial cells, resulting in a decrease in reduced nitrite/nitrate concentrations. Peripheral vascular disorders caused by arsenic may be attributable in part to impairment of NO production in vivo.

A potential mechanism for the impairment of nitric oxide formation caused by prolonged oral exposure to arsenate in rabbits

We have recently found evidence for impairment of nitric oxide (NO) formation and induction of oxidative stress in residents of an endemic area of chronic arsenic poisoning in Inner Mongolia, China. To investigate the underlying mechanisms responsible for these phenomena, a subchronic animal experiment was conducted using male New Zealand White rabbits. After 18 weeks of continuous exposure of rabbits to 5 mg/l of arsenate in drinking water, a significant decrease in systemic NO production occurred, as shown by significantly reduced plasma NO metabolites levels (76% of control) and a tendency towards decreased serum cGMP levels (81.4% of control). On the other hand, increased oxidative stress, as shown by significantly increased urinary hydrogen peroxide (H(2)O(2)) (120% of control), was observed in arsenate-exposed rabbits. In additional experiments measuring aortic tension, the addition of either the calcium ionophore A23187 or acethylcholine (ACh) induced a transient vasoconstriction of aortic rings prepared from arsenate-exposed rabbits, but not in those prepared from control animals. This calcium-dependent contractility action observed in aorta rings from arsenate-exposed rabbits was markedly attenuated by the superoxide (O2(.-)) scavenging enzyme Cu, Zn-SOD, as well as diphenyleneiodonium (DPI) or N(G)-nitro-L-arginine methyl ester (L-NAME), which are inhibitors for nitric oxide synthase (NOS). However, the cyclooxygenase inhibitor indomethacin or the xanthine oxidase blocker allopurinol had no effect on this vasoconstriction. These results suggest that arsenate-mediated reduction of systemic NO may be associated with the enzymatic uncoupling reaction of NOS with a subsequent enhancement of reactive oxygen species such as O2(.-), an endothelium-derived vasoconstricting factor. Furthermore, hepatic levels of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH(4)), a cofactor for NOS, were markedly reduced in arsenate-exposed rabbits to 62% of control, while no significant change occurred in cardiac L-arginine levels. These results suggest that prolonged exposure of rabbits to oral arsenate may impair the bioavailability of BH(4) in endothelial cells and, as a consequence, disrupt the balance between NO and O2(.-) produced from endothelial NOS, such that enhanced free radicals are produced at the expense of NO.

Diphenylarsinic acid poisoning from chemical weapons in Kamisu, Japan

We noted a new clinical syndrome with prominent cerebellar symptoms in apartment building residents in Kamisu, Japan. The well that provided drinking water contained diphenylarsinic acid, a degradation product of diphenylcyanoarsine or diphenylchloroarsine, which were developed for use as chemical weapons, inducing severe vomiting and sneezing. Characteristics of diphenylarsinic acid poisoning include brainstem–cerebellar and cerebral symptoms. Mental retardation associated with brain atrophy in magnetic resonance images was evident in some infants. We must be vigilant to prevent or minimize the effects of further diphenylarsinic acid poisoning in Japan or elsewhere. Ann Neurol 2004;56:741–745

Impairment of spermatogenesis in rats by methylmercury: involvement of stage- and cell- specific germ cell apoptosis.

Methylmercury has been shown to affect the male reproductive organs. However, the specific mode of impairment of spermatogenesis during methylmercury exposure remains unknown. In this study, we characterized the induction of germ cell apoptosis and reproductive toxicity in Wistar male rats that had been exposed to methylmercuric chloride (MMC). Subcutaneous injection of MMC at a dose of 10 mg/kg per day for 8 days resulted in a 28% testicular weight loss at 14 days after the first injection. In addition, the ventral and dorso-lateral prostatic lobes showed a 65 and 52% decrease, respectively, at 14 days, although no effects were observed in the epididymis. Sperm production also was suppressed by the administration of MMC. After exposure to MMC, fragmentation of testicular DNA was found to be increased at 3 days after the first injection, with a 20-fold increase over control levels at 14 days. In situ detection of apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) staining revealed that spermatocytes and spermatids at stages VII-VIII and IX-XI, respectively, steps which are considered to be highly sensitive to testosterone, were the major cell types affected. Consequently, a marked cell loss in elongated spermatids at stages XII-XIV and I was observed at 14 days. In addition, plasma testosterone levels were reduced at 6 days after exposure to MMC, and remained at approximately 20% of control levels during the 14-day observation period. Our results suggest that methylmercury impairs spermatogenesis by germ cell deletion via cell- and stage- specific apoptosis.

Detection of sulfur-containing compounds in control and cadmium-exposed rat organs by high-performance liquid chromatography-vacuum-ultraviolet inductively coupled plasma-atomic emission spectrometry (HPLC-ICP)

Sulfur-containing compounds in biological samples were separated by high-performance gel permeation chromatography and detected with a vacuum-ultraviolet inductively coupled plasma-atomic emission spectrometer. Distribution profiles of sulfur in the supernatants of liver, kidney, spleen, lung, and pancreas of control and cadmium-exposed rats were determined along with cadmium, copper, iron, phosphorus, and zinc profiles. Changes in sulfur distribution were induced by cadmium exposure not only in the metallothionein fraction, but also in the high-molecular-weight protein fraction, indicating the effect of cadmium exposure on diverse endogenous sulfur-containing compounds. Glutathione and taurine also were detected simultaneously as distinct peaks.

Gene deficiency of glutathione S-transferase μ isoform associated with susceptibility to lung cancer in a Chinese population

Increased lung cancer risk associated with genetic polymorphism of glutathione S-transferase (GST, EC 2.5.1.18) isozyme mu was examined in a Chinese population. A significantly higher proportion in lung cancer patients showed GST mu deficiency compared with control group (71.0% vs. 51.1%, P < 0.005). Although the susceptibility to lung cancer showing gene deletion for GST mu isoform in non-smoking group is not significantly different from that in smoking group, a great number of individuals with gene deletion was found among cancer patients who are less than 50 years old. The pathology of lung tumors related to that lack of class mu isoform which occurred most frequently in patients with small cell carcinomas. Thus, present data further support that sensitivity to chemical toxins and pulmonary carcinogens may be affected by GST mu isoform polymorphism.

Mercaptalbumin as a selective cadmium-binding protein in rat serum

Cadmium-binding proteins in blood serum were determined on a gel permeation column by high-performance liquid chromatography-inductively coupled argon plasma-atomic emission spectrometry. Cadmium chloride was administered iv to female rats of the Wistar strain in a single dose of 0.4 mg Cd/kg body wt and the rats were killed 1, 2, 3, 5, 10, 20, and 30 min after the injection. The blood serum was separated on an Asahipak GS-520 column and cadmium concentration in the eluate was monitored continuously along with sulfur, zinc, copper, iron, and phosphorus concentrations. Cadmium was selectively bound to mercaptalbumin and the metal was eliminated from blood serum within 30 min in a biphasic mode. Addition in vitro of cadmium chloride into fresh blood serum revealed that cadmium is bound selectively to mercaptalbumin up to approximately 14 micrograms Cd/ml serum. Excess cadmium in blood serum was found in the lower molecular weight fraction. Nonmercaptalbumin produced by oxidative disulfide bond formation between albumin and glutathione or cysteine was not able to bind cadmium. Sulfur and other element profiles were helpful in characterizing metal-binding proteins.

Selective induction of apoptosis of renal proximal tubular cells caused by inorganic mercury in vivo

A recent notion, that a variety of toxicants causing necrosis can lead to apoptosis as well, has been demonstrated with cultured cells, but not with in an vivo system. In the present study, we examined the induction of both apoptosis and necrosis in the kidneys of Wistar rats exposed to mercuric chloride (HgCl(2)). A single injection of HgCl(2) to rats at a dose of 4 mg/kg resulted in an increase in the renal DNA fragmentation evaluated as an occurrence of apoptosis, prior to urinary excretion of alkaline phosphatase (ALP) and renal morphological changes assessed as necrotic phenomena. The mercury-promoted DNA fragmentation was induced in a dose-dependent manner. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and morphological observation of the nuclei revealed that apoptotic cells caused by HgCl(2) were predominantly found in the proximal tubules, but not in the distal tubules, glomeruli or medullary tubules. When we confirmed the proximal tubular-selective apoptosis by inorganic mercury with a combined technique of TUNEL staining with synchrotron radiation X-ray fluorescence (SR-XRF) imaging, it was shown that the apoptotic cells localized in the proximal tubules did contain higher level of mercury. Thus these results indicate that the proximal tubular cells-dominant site-specific distribution of mercury appears to be associated with induction of renal apoptosis and necrosis.

ζ-Crystallin catalyzes the reductive activation of 2,4,6-trinitrotoluene to generate reactive oxygen species: a proposed mechanism for the induction of cataracts

Exposure to 2,4,6‐trinitrotoluene (TNT) has been shown to cause induction of cataract in which oxidative stress plays a critical role. From bovine lens we purified to homogeneity and identified an enzyme that catalyzes the reduction of TNT, resulting in the production of reactive oxygen species. The final preparation of TNT reductase showed a single band with a subunit molecular weight of 38 kDa on SDS–PAGE. Sequence data from peptides obtained by digestion with lysylendopeptidase Achromobacter protease I (API) revealed that TNT reductase is identical to ζ‐crystallin. Superoxide anions were formed during reduction of TNT by ζ‐crystallin, though negligible enzyme activity or protein content for superoxide dismutase, a superoxide scavenging enzyme, was found in the lens. Thus, the present results suggest that the induction of cataracts by TNT may be associated with increased oxidative stress, as a result of reductive activation of TNT generating superoxide anions, there being minimal antioxidant enzyme activity for defense against reactive oxygen species exogenously produced in the lens.