Shino Homma-Takeda

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.

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.

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.

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.

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.

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.

Alterations in Superoxide Dismutase Isozymes by Methylmercury

A study of alterations in two mammalian superoxide dismutases (SODs), Cu,Zn-SOD and Mn-SOD, caused by methylmercury has been reviewed. Mechanisms for the isozyme-selective decrease in MnSOD activity by the organometallic compound observed in vivo have been extensively examined by experiments with purified enzyme preparations.

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.

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.

Exposure of Rat to Inorganic Mercury Induces DNA Fragmentation Responsible for Apoptosis in Kidney

. Recently, toxicant-induced apoptosis has been reported for toxic metals, such as chromium3) and methylmercury4) in cultured cells. Little information, however, about metal-promoted apoptosis in vivo has been reported and the question whether apoptotic phenomena occur during exposure of animals to mercury still remains unclear. In the study reported here, we have extended our findings to mercury nephrotoxicity and show here that administration of mercury to rats results in DNA fragmentation characteristic of apoptosis in an in vivo kidney system.