Microarray analysis of 6-mercaptopurine-induced-toxicity-related genes and microRNAs in the rat placenta.

MicroRNAs (miRNAs) are small single-stranded RNAs of 19-25 nucleotides and are important in posttranscriptional regulation of genes. Recently, the role of miRNAs in toxicity incidence is reported to be a regulator of key-stopper of gene expression, however the detailed mechanism of miRNAs is not well known yet. 6-Mercaptopurine (6-MP), the anti-leukemic and immunosuppressive drug, produced teratogenicity and pregnancy loss. We focused on the placenta to evaluate toxicity in embryo/fetal development produced by 6-MP treatment. MiRNA expression in the placenta was analyzed by miRNA microarray. Fifteen miRNAs were upregulated on GD13 and 5 miRNAs were downregulated on GD15 in 6-MP treatment rat placentas. Some miRNAs may have functions in apoptosis (miR-195, miR-21, miR-29c and miR-34a), inflammation (miR-146b), and ischemia (miR-144 and miR-451). In the maternal plasma, expression of miR-144 was significantly reduced by 6-MP treatment when examined by real-time RT-PCR. We determined toxicity-related gene expression in the rat placenta. Gene expression analysis was carried out by DNA oligo microarray using rat placenta total RNAs. Compared between predicted targets of miRNAs and microarray data in 6-MP-treated rat placenta, expressions of hormone receptor genes (estrogen receptor 1; Esr1, progesterone receptor; Pgr, and prolactin receptor; Prlr), xanthine oxidase (Xdh), Slc38a5 and Phlda2 genes were changed. The histopathologically found increase in trophoblastic giant cells and reduced placental growth by 6-MP treatment were well correlated to these gene expressions. These data suggest that some miRNAs may link to toxicological reactions in 6-MP-induced placental toxicity.

6-Mercaptopurine-induced histopathological changes and xanthine oxidase expression in rat placenta.

The placenta secures the embryo and fetus to the endometrium and releases a variety of steroid and peptide hormones that convert the physiology of a female to that of a pregnant female. Chemical-induced alteration or deviation of placental function in the maternal and extraembryonic tissue can ultimately lead to pregnancy loss, congenital malformation and fetal death. The 6-mercaptopurine (6-MP), an anti-leukemic drug, is known to produce undesired effects on some organs, then the placenta/embryo toxicity of 6-MP was investigated in pregnant rats given 60 mg/kg with two intraperitoneal injections on gestation days (GD) 11 and 12. The rats were sacrificed and their placentas were collected on GD13 or 15. On GD15 small and limb-defected embryos were found in the 6-MP-treated rats. Placental weights were significantly reduced on GD15, as well as a reduced number of cells was detected in the labyrinth zone with both the labyrinth and basal zones having thinned. Cleaved caspase-3-positive cells increased in number in the labyrinth zone, while in the basal zone, glycogen cells reduced with cytolysis. The number of spongiotrophoblasts and trophoblastic giant cells also increased by 6-MP treatment. The 6-MP-treatment resulted in the increased xanthine oxidase (Xdh) expression in the placenta, which gene is related to the ischemic condition of tissues. These data suggest that apoptosis of the labyrinth zone cells may lead to decreased materno-fetal exchange. Moreover, subsequent ischemia in the placental tissue may occur and induce Xdh expression.

Questionnaire results of hyperbaric oxygen therapy for acute carbon monoxide poisoning in Japan.

Carbon monoxide (CO) poisoning results in not only severe psychoneurological disorders, but can also cause secondary delayed psychoneurological disorders. Therefore, timely and appropriate treatment in the acute stage is crucial to prevent such direct neurological damage and secondary disorders. However, various conflicting results have been reported in studies of CO poisoning treatment, and the efficacy of hyperbaric oxygen therapy (HBO2T) for CO poisoning has not been established. This retrospective multi-institutional study was performed by the questionnaire in 1667 cases of acute CO poisoning in Japan. The effectiveness of HBO2T for CO poisoning was evaluated based on prognoses in cases and various classes of hospital based on the grade of their positive stance regarding HBO2T. The results showed that the prognosis in the group treated with HBOT was significantly better than that in the group treated with normobaric oxygen therapy (NBO2T) (P < 0.01), thus confirming the effectiveness of HBO2T for CO poisoning. Furthermore, while hospitals were separated into three groups according to their indication criteria for HBO2T, the ineffective ratio of NBO2T was dependent on the indication criteria, even though the effective ratio of HBO2T was the same in all three groups. In conclusion, a retrospective multi-institutional study showed that HBO2T is an effective form of therapy for CO poisoning.

Exogenously-added copper/zinc superoxide dismutase rescues damage of endothelial cells from lethal irradiation.

The vascular endothelium is important for the early and late effects observed in lethally irradiated tissue and organs. We examined the effects of exogenously added superoxide dismutase on cell survival and angiogenesis in lethally irradiated human primary umbilical vein endothelial cells. Cell survival was significantly improved in superoxide dismutase-treated cells; the addition of superoxide dismutase to cells after irradiation was also effective for increased survival, as it was before irradiation. Moreover, treatment of cells with superoxide dismutase enhanced the phosphorylation of mitogen-activated protein/extracellular signal-regulated kinase/extracellular signal regulated kinases 1 and 2 in human primary umbilical vein endothelial cells. The addition of superoxide dismutase to cells after irradiation attenuated the reduction of angiogenesis by irradiation, and inhibition of the mitogen-activated protein/extracellular signal-regulated kinase/extracellular signal regulated kinases signaling pathway abrogated the rescue effect of superoxide dismutase. Our results suggest that superoxide dismutase rescues human primary umbilical vein endothelial cells from endothelial dysfunction caused by irradiation via a pathway requiring activation of mitogen-activated protein/extracellular signal-regulated kinase/extracellular signal regulated kinases 1 and 2.

MicroRNAs expression in the ethylene glycol monomethyl ether-induced testicular lesion.

Ethylene glycol monomethyl ether (EGME) induces testicular lesion in rats and human. To investigate miRNAs expression in EGME testicular lesion, miRNA array assay and real-time RT-PCR analysis were conducted by using testis in rats treated with 50 and 2,000 mg/kg EGME for 6 and 24 hr. The expression of corresponding target gene for miRNAs was also examined. At 50 mg/kg, there were no changes in the gene expression and histopathological examination. At 2,000 mg/kg, slight decrease of phacytene spermatocytes with cell shrinkage and nucleus pyknosis at 6 hr and remarkable decrease (or cell death) of phacytene spermatocytes with Sertoli cell vacuolation at 24 hr were observed. After 24 hr, miR-449a and miR-92a decreased obviously and, miR-320, miR-134 and miR-188 increased, while only miR-760-5p increased after 6 hr. Above these miRNAs are reported to have an important role for spermatogenesis. The gene expression of Bcl-2, target for miR-449a, increased and therefore it is considered anti-apoptotic reaction has started in this stage. The expression of high mobility group AT-hook 2 (target of miR-92a) which regulates histone structure, was increased. Furthermore, histone deacethylase 4, targets for miR-320, was also affected. Above prohibiting apoptosis or activating epigenetic genes might be protective reaction to spermatocytes death under the miRNAs regulation in EGME testicular lesion.

Changes in leukocyte migration during carbonic anhydrase activity inhibition.

Experiment 1 evaluated changes in leukocyte migration during acetazolamide (AZ) inhibition of carbonic anhydrase activity in leukocytes. AZ induced changes in the intracellular calcium concentration, and extracellular calcium is thought to be a factor inducing an increase in leukocyte migration. Next, Experiment 2 determined whether extracellular calcium concentration was a primary factor influencing leukocyte migration in the absence of AZ. The distance of leukocyte migration increased in a dose-dependent manner with AZ despite the presence of IL-8 or LPS in Experiment 1. The extracellular calcium concentration used in the present study had no influence on the distance in leukocyte migration in Experiment 2. The distance of leukocyte migration showed a tendency to increase in a dose-dependent manner with LPS concentration. In conclusion, AZ may stimulate leukocyte migration due to its participation in the regulation of intracellular pH controlled by CA activity without an effect of low extracellular calcium concentration. In addition, AZ was thus suggested to possibly have an anti-inflammatory effect in supporting leukocyte migration during inflammatory reactions.

Apoptosis signal-regulating kinase 1 is crucial for oxidative stress-induced but not for osmotic stress-induced hepatocyte cell death.

AIMS: In this study, we investigated the involvement of apoptosis signal-regulating kinase 1 (ASK1) in oxidative stress and osmotic stress-induced hepatocyte death. MAIN METHODS: Activation of ASK1-JNK/p38 cascade and resulting cell death induced by oxidative and osmotic stress was investigated by Western immunoblot analysis and cell toxicity assay using human hepatoma cell lines, Huh7 expressing high level of ASK1 and HepG2 cells expressing low level of ASK1. Gene knock-down of ASK1 using shRNA against ASK1 was conducted using mouse hepatocyte cell line, AML12. KEY FINDINGS: Activation of ASK1-JNK/p38 cascade and cell death in Huh7 expressing high level of ASK1 was markedly induced by the oxidative stress. HepG2 expressing low level of ASK1 was resistant to oxidative stress while cell death induced by osmotic stress was comparable between Huh7 and HepG2 cells. Although the phosphorylation of ASK1 was not observed by osmotic stress, the phosphorylation of p38 and JNK and resulting cell death was induced in both cell lines. The phosphorylation of ASK1 and p38/JNK in the mouse primary hepatocyte were also increased by oxidative stress. Knock-down of ASK1 mRNA in AML12 in vitro significantly reduced oxidative stress-induced cell death, however, knock-down of ASK1 in cells did not affect the osmotic stress-induced cell death. SIGNIFICANCE: This study revealed that ASK1 regulates oxidative stress- but not osmotic stress-induced hepatocyte death, suggesting ASK1 plays a critical role in oxidative-stress induced hepatocyte death. These results raise the possibility that an ASK1 may be a promising therapeutic target for liver diseases caused by oxidative stress.

Correlation between projection of the ear, the inferior crus, and the antihelical body: analysis based on computed tomography.

We investigated the correlation between projection of the ear and the antihelical folding angle to clarify which portion of the antihelix should be corrected in reconstruction of prominent ears using computed tomograms of 15 ears in 11 patients with fractures of the facial bones. The angle of the scaphotriangular fossa indicating the superior crus, cymba conchae-triangular fossa angle indicating the inferior crus, and the scaphoconchal angle indicating the antihelical body were measured. There was no relation between the cranioauricular angle and the angle of the scaphotriangular fossa. However, there were significant relations between the cranioauricular angle and the cymba conchae-triangular fossa angle, and the cranioauricular and scaphoconchal angles, which suggests that emphasis should be placed on reconstruction of the inferior crus and antihelical body when prominent ears are being corrected.

Seasonal changes in distribution and abundance of euphausiids in the coastal area of north-eastern Japan.

Seasonal changes in distribution and abundance of euphausiids off south-eastern Hokkaido (41°-43°N), Sanriku (38°-41°N), and Joban (36°-38°N) were investigated using cylindrical-conical nets every two months from March 1997 to February 1998. Twenty-six species of seven genera of euphausiids occurred during the survey. Among them, subarctic-transitional Euphausia pacifica was the most abundant throughout the year in coastal waters, as their relative contribution to the total abundance of euphausiids was 89-92%. This species occurred in each coastal water throughout the survey and was abundant from winter to early summer (February-June) off Sanriku and Joban and in autumn in south-eastern Hokkaido. Thysanoessa inspinata occurred off south-eastern Hokkaido and Sanriku throughout the survey, mainly in spring (April) but rarely occurred off Joban. Three other subarctic Thysanoessa species occurred mainly off south-eastern Hokkaido from winter to spring. Conversely, warm- and transitional-water epipelagic species occurred exclusively off Sanriku and Joban in autumn. The characteristics of seasonal distributional patterns of euphausiids are discussed in relation to the spatial and temporal changes of oceanographic conditions and several predators off north-eastern Japan.

[A study on the factors influencing the anxiety of family members in the emergency department].

The purpose of this study was to determine the factors which affect anxiety of family members in the emergency department (ED). 174 family members of patients participated in this study. The age of family members was a mean of 43.1 (range: 20 to 84) years and 59.8% of them were women. The informations were obtained from a questionnaire filled out by the family members when they were waiting during examination and treatment of the patients. In this study, we divided the factors that influence the anxiety of family members into 4 categories; demographic factors, the family's individual factors, factors associated illness, and environmental factors in the ED. Multiple regression analysis with SPSS was used to identify the variables contributing to the variance in anxiety. We used the State Anxiety Inventory (S-STAI) to measure anxiety. As a result, 8 variables involving in severity of illness, situation in the emergency room, disagreement between perceived severity of illness and actual severity of illness, having symptoms of trauma, neurological, heart, and respiratory problems, waiting time, family needs, naturally anxious personality and a first visit patient were identified as significant predictors of anxiety. These variables accounted for 46.9% of total variance. These results suggest that nurses need more interaction with family members to reduce their anxiety.

Determination of methamphetamine and amphetamine in abusers' plasma and hair samples with HPLC-FL.

This paper describes highly sensitive HPLC methods for the determination of amphetamine (AP) and methamphetamine (MP) in abusers' plasma and hair samples. AP and MP were derivatized with the fluorescent reagent, DIB-Cl, to yield a highly fluorescent DIB-derivatives of AP and MP, which were then analyzed by HPLC with fluorescence detection at excitation and emission wavelengths of 325 and 430 nm, respectively. The separation was achieved on an ODS column with isocratic mobile phases composed of acetoniltrile and citrate buffer (55:45, v/v) for plasma samples and of acetonitrile-methanol-citrate buffer (45:20:37.5, v/v/v) for hair samples. The limits of detection were less than 0.87 ng/mL and 0.12 ng/mg in plasma and hair samples, respectively, for both AP and MP. The methods were then applied to the determination of MP and its metabolite AP in plasma obtained from two cases of illegally ingested MP and in one of the cases' hair received later. Case I was treated with dialysis; samples before and after dialysis were analyzed by the described method. After dialysis for 5 h, the total plasma levels of AP and MP decreased from 720 to 190 ng/mL. For case II, MP and AP levels were monitored for 3 days after digestion. Total plasma levels decreased from 57 ng/mL in the day of digestion to 11 ng/mL after 3 days. In hair samples, AP and MP could also be detected in very low concentrations.

[A case of successfully treated nifedipine-poisoning].

Calcium antagonists have been prescribed for treatment of hypertension and several other diseases, and the incidence of poisoning involving these agents is increasing. We encountered and successfully treated a case of nifedipine poisoning. The patient was a 52-year-old man who ingested 76 tablets of nifedipine 20 mg while drinking alcohol. He was brought to a clinic and transferred to our emergency department. Since systolic blood pressure on arrival was 110 mmHg, primary care involved gastric lavage, infusion of lactated Ringer solution, and administration of activated charcoal and cathartics. Hypotension subsequently developed and continuous infusion of dobutamine was initiated. Arrhythmia did not appear during the course of treatment, and the patient was discharged after four days.

Analysis of ischemic brain damage in cases of acute subdural hematomas.

BACKGROUND: Ischemic damage of the brain is one of the most important factors for the sequelae of acute subdural hematomas (ASDHs). However, ischemic damage is infrequently addressed in a systematic manner in the clinical setting. METHODS: The analysis of ischemic brain damage was performed based on serial computed tomography (CT) scans in 80 patients with traumatic ASDHs. Single photon emission computed tomography (SPECT) for regional blood flow and/or magnetic resonance imaging (MRI) were also performed. RESULTS: Follow-up CT scans showed ischemic brain damage in 19 patients and no significant damage in 35 patients. The remaining 26 patients progressively deteriorated to the point of brain death. The ischemic brain damage was seen most frequently in the territory of the anterior cerebral artery (13 cases), followed by the territory of the posterior cerebral artery (12 cases). The ischemic damages in the pallidum, the hypothalamus and the thalamus were demonstrated in 4, 8, and 4 cases, respectively. The ischemic damage in the underlying brain that was probably because of the direct compression of the hematoma was seen in only two cases. CONCLUSIONS: Most of the ischemic brain damage noted in this study was because of arterial compression secondary to the brain shift and brain herniation, rather than the direct effect of the hematoma upon the underlying brain. Ischemic brain damage adversely affects outcome morbidity, and the difficulty in preventing ischemic damage in cases with marked brain shift leads to poor outcome in patients with ASDHs.

Regulation of hemoglobin affinity for oxygen by carbonic anhydrase.

We studied the effect on hemoglobin (Hb)-oxygen affinity induced by changes in carbonic anhydrase (CA) activity. Oxygen partial pressure at the 50% saturation of Hb (P(50)) in human blood was measured as CA activity was inhibited to varying degrees with acetazolamide (AZ; 100 and 200 microg/mL). Transient but significant change in P(50) was observed when AZ was administered and the CO(2) concentration was changed from 10% to 5%. Finally, the differences induced with AZ were attenuated when the blood sample was subjected to 4 hours of tonometry. The findings in this study could be accounted for by reduced velocity of pH changes caused by the inhibition of CA by AZ. We conclude that CA can change Hb's affinity for oxygen by controlling the movement of CO(2) gas between air and liquid compartments.

Detection of acetazolamide-induced increase in organ blood flow in rabbits by laser flowmetry.

PURPOSE: We investigated the mechanism by which inhibition of carbonic anhydrase (CA) increases organ blood flow. METHODS: Regional blood flow (rBF) in white rabbits anesthetized with ketamine/urethane was measured in the kidney, liver, stomach wall, and abdominal muscle by means of laser blood flow probes. Data obtained from rabbits receiving acetazolamide (AZ) to inhibit CA were compared with those obtained from rabbits ventilated with air containing increased concentrations of CO2. RESULTS: Systolic blood pressure, body temperature, hemoglobin, and base excess were unaffected by either treatment. Inhalation of CO2 increased blood flow in all organs tested as well as the cardiac output and PCO2 but decreased pH. Inhibition of CA by AZ administration increased the rBF only in the liver and kidney and did not increase cardiac output or decrease pH. CONCLUSION: Administration of AZ increased rBF in the tissues and organs that contained large amounts of CA without increasing the cardiac output or decreasing the pH, which suggests a direct local effect. A differential sensitivity to the retention of CO2 is suggested as a possible mechanism of the selectivity of the increase in rBF.