BYSTANDER WI-38 CELLS MODULATE DNA DOUBLE-STRAND BREAK REPAIR IN MICROBEAM-TARGETED A549 CELLS THROUGH GAP JUNCTION INTERCELLULAR COMMUNICATION.

Bi-directional signaling involved in radiation-induced bystander effect (RIBE) between irradiated carcinoma cells and their surrounding non-irradiated normal cells is relevant to radiation cancer therapy. Using the SPICE-NIRS microbeam, we delivered 500 protons to A549-GFP lung carcinoma cells, stably expressing H2B-GFP, which were co-cultured with normal WI-38 cells. The level of γ-H2AX, a marker for DNA double-strand breaks (DSB), was subsequently measured up to 24-h post-irradiation in both targeted and bystander cells. As a result, inhibition of gap junction intercellular communication (GJIC) attenuated DSB repair in targeted A549-GFP cells, and suppressed RIBE in bystander WI-38 cells but not in distant A549-GFP cells. This suggests that GJIC plays a two-way role through propagating DNA damage effect between carcinoma to normal cells and reversing the bystander signaling, also called 'rescue effect' from bystander cells to irradiated cells, to enhance the DSB repair in targeted cells.

IMAGING URANIUM DISTRIBUTION ON RAT KIDNEY SECTIONS THROUGH DETECTION OF ALPHA TRACKS USING CR-39 PLASTIC NUCLEAR TRACK DETECTOR.

Uranium is renowned as a global contaminant, and attracts major concern with regards to the health risks involved because its nephrotoxicity. This paper discusses the development of a simple method to identify accumulated regions or localized sites of uranium within kidneys using the CR-39 plastic nuclear track detector. To demonstrate the proposed method, renal cryo-sections (5 µm-t) from Wistar male rats, subcutaneously administered with uranyl acetate (2 mg/kg), were prepared on day one after administration. Concerned sections were subsequently placed on CR-39, stored for 1.25 years, and then etched in a 7 M NaOH solution at 70°C for 3 h. α-tracks were then detected in the form of etch pits, corresponding to uranium, and also the tissue shape and structure were transferred as a roughness on the surface of CR-39. As observed, the proposed method served to facilitate simultaneous detection and identification of localized regions of uranium accumulation within kidneys.

Biokinetics and dose estimation of 65Zn in the salivary gland and male reproductive organs.

ICRP is revising its recommendations for radiological protection and has added salivary and secretory glands as new target organs. However, little information is available on the distributions of radionuclides in the salivary gland, secretory glands and male reproductive organs. This study deals with the distribution of 65Zn in the salivary gland and male reproductive organs as a function of time after a single intravenous and oral administration. For the study, 64 Wistar strain male rats, eight weeks of age were used. The rats were periodically sacrificed, the liver, kidney, spleen, pancreas, thymus, salivary gland, testis, epididymitis and prostate gland sampled and the radioactivity of these organs measured with an NaI scintillation counter. The relative concentration of 65Zn was highest in the prostate gland. We estimated the radiation dose in humans using rat data for the salivary and secretory glands as well as reproductive organs after intake of 65Zn.

Biokinetics of radiotellurium in rats.

Radiotellurium is present in the environment primarily due to its release during nuclear reactor accidents. Little is known of tellurium metabolism in juveniles, although the element is relatively abundant and has a number of industrial uses. A biokinetic study of radiotellurium in rats was performed using gamma-ray counting. Wistar strain rats were used to determine the uptake of H2(123m)TeO3 by the whole-body retention of juvenile rats and the conceptus in relation to its gestational stages, by measurements in the placenta, fetal membranes, fetal fluid and fetus. The whole-body retention of 123mTe in juvenile rats was higher than that of adult rats. The relative concentration in the placenta and fetal membranes was higher than in the fetus. No activity was observed in the fetal fluid. These results indicate that the placenta and fetal membranes play significant roles as barriers to the transfer of 123mTe into the fetus. The ratio, relative concentration in fetus/relative concentration in mother (C(F)/C(M)), was calculated. The C(F)/C(M) ratio was dependent on the stage of gestation and ranged from 0.2 to 0.5. A little 123mTe was transferred to the suckling rats through the mother's milk when the isotope was administered intravenously to the mother.

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.

Enzyme activity and protein content of superoxide dismutase isozymes in human renal cell carcinoma.

Alterations in renal superoxide dismutase (SOD) isozymes were examined in cancerous tissues of human renal cell carcinoma and the corresponding non-cancerous renal tissues. Cu,Zn-SOD activities in cancerous tissues were lower than those in normal tissues. Mn-SOD activities were varied in the cases examined, whereas no significant difference between cancerous and normal tissues was observed for Mn- or total-SOD activities. Immunoblot analysis showed that the loss in enzyme activity in cancerous tissue was greater than the decrease in protein content for either isozyme. The selective decrease in Cu,Zn-SOD activities in cancerous tissue observed in this study suggests that the cytoplasmic defense against free radical damage appears to be reduced in renal cell carcinoma.

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.

Inhibition of nitric oxide formation and superoxide generation during reduction of LY83583 by neuronal nitric oxide synthase.

6-Anilino-5,8-quinolinedione (LY83583) has been widely used as an agent to reduce levels of nitric oxide (NO)-dependent cGMP in tissues. We report here that suppression of NO formation and production of superoxide during enzymatic reduction of LY83583 by neuronal NO synthase appeared to be potentially involved in the pharmacological action caused by LY83583. LY83583 suppressed neuronal NO synthase activity of 20,000 x g rat cerebellar supernatant preparation in a concentration-dependent manner (IC50 value = 12.9 microM). A kinetic study revealed that LY83583 is a competitive inhibitor with respect to NADPH, with a Ki value of 2.57 microM. With purified neuronal NO synthase it was found that LY83583 was a potent inhibitor of NO formation by the enzyme and served as efficient substrate for reduction with a specific activity of 173 nmol of NADPH oxidized per mg of protein per minute. The reductase activity was stimulated about 19.8-fold by addition of CaCl2/calmodulin, indicating that the presence of CaCl2/calmodulin is essential to express maximal activity of LY83583 reduction. Although LY83583 was a good substrate for both NADPH-cytochrome P450 reductase (P450 reductase) and DT-diaphorase, these flavin enzymes-catalyzed reductions of LY83583 were less than the neuronal NO synthase-mediated reduction in the presence of CaCl2/calmodulin. Enzymatic generation of superoxide during reduction of LY83583 by neuronal NO synthase, P450 reductase or DT-diaphorase was confirmed by electron spin resonance (ESR) experiments. Thus the present results indicate that a benzoquinone derivative LY83583 appears to interact with the P450 reductase domain on neuronal NO synthase, resulting in inhibition of NO formation and superoxide generation, which is involved in suppression of intracellular cGMP content.

Apoptosis in rat renal proximal tubular cells induced by cadmium.

Cadmium chloride can induce DNA fragmentation, a biochemical characteristic of apoptosis in renal epithelial LLC-PK, cells. This study was extended to determine the in vivo effects of this heavy metal on apoptosis. Nephrotoxicity was induced by a single intravenous administration of cadmium-metallothionein (0.15 mg metallothionein-bound cadmium/kg body weight) to male Jcl:Wistar rats. DNA fragmentation was seen in the kidney 12 h after injection of cadmium-metallothionein without a concurrent release of lactate dehydrogenase in urine. Cycloheximide (3 mg/kg) inhibited cadmium-induced DNA fragmentation, suggesting that protein synthesis might be required for the induction of cell death by this metal. Apoptotic cells were identified in proximal tubular cells by in situ DNA 3'-end labeling. Furthermore, chromatin condensation in the apoptotic population of renal proximal tubular cells was noted. Data thus suggest that cadmium produces biochemical and morphological alterations in kidney, which are characteristic features seen in apoptosis.

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.

Inhibition of nitric oxide formation by neuronal nitric oxide synthase by quinones: nitric oxide synthase as a quinone reductase.

Inhibitory action of a variety of quinoid compounds on neuronal nitric oxide synthase (nNOS) activity was examined with a 20000g rat cerebellar supernatant preparation and purified nNOS. The inhibition of citrulline formation from l-arginine by quinones, which exhibit one-electron reduction potentials (E17) ranging between -240 and -100 mV, increased at a more positive one-electron reduction potential, suggesting that quinone appears to act as an electron acceptor for nNOS. Among the quinones tested, 9,10-phenanthraquinone (PQ), corresponding to an E17 value of -124 mV, exhibited the most potent inhibiton of citrulline formation (IC50 value = 10 microM). A kinetic study revealed that PQ is a competitive inhibitor with respect to NADPH, with a Ki value of 0.38 +/- 0.12 microM, and a noncompetitive inhibitor with respect to l-arginine, with a Ki value of 9.63 +/- 0.20 microM. Partial purification of the enzymes which are responsible for reducing PQ in 20000g supernatant of rat cerebellum by anion-exchange column chromatography indicated that one catalyst for PQ reduction was nNOS. Reductase activity of PQ by purified nNOS required CaCl2/calmodulin and was markedly suppressed by the flavoprotein inhibitor diphenyleneiodonium but not by l-nitroarginine which is a specific inhibitor for NO formation. nNOS effectively reduced the quinones as well as PQ causing a marked decrease in the production of NO from l-arginine, while 1, 4-benzoquinone, 9,10-anthraquinone, mitomycin C, and lapachol, which show negligible inhibitory action on nNOS activity, were poor substrates for the enzyme on reduction. These results indicate that PQ and other quinones used in the present study interact with the NADPH-cytochrome P450 reductase domain on nNOS and thus probably inhibit NO formation by shunting electrons away from the normal catalytic pathway. Therefore, our study suggests that quinones could possibly affect NO-dependent physiological and/or pathophysiological actions in vivo.

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.

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.