[Correlation between balloon volume and Meckel's cave size and its influence of percutaneous microballoon compression for trigeminal neuralgia].

Objective: To investigate the correlation between balloon volume and Meckel's cave size during percutaneous puncture microballoon compression (PMC) for trigeminal neuralgia and the influence of the compression coefficient (the ratio of balloon volume/Meckel's cave size) on the prognosis. Methods: Seventy-two patients (28 males and 44 females) aged (62±11) years who underwent PMC under general anesthesia for trigeminal neuralgia in the First Affiliated Hospital of Zhengzhou University from February 2018 to October 2020 were retrospectively collected. All patients underwent preoperative cranial magnetic resonance imaging (MRI) to measure Meckel's cave size, intraoperative balloon volume was recorded, and the compression coefficient was calculated. Follow-up visits were performed preoperatively (T0) and 1 d (T1), 1 month (T2), 3 months (T3), and 6 months (T4) postoperatively, either in the outpatient clinic or by telephone, and the Barrow Neurological Institute pain scale (BNI-P) score, the Barrow Neurological Institute facial numbness (BNI-N) score and the occurrence of complications were recorded and compared at each time point. Patients were divided into 3 groups according to different prognoses: patients in group A (n=48) were with no recurrence of pain and mild facial numbness, patients in group B (n=19) were with no recurrence of pain but severe facial numbness, while those in group C (n=5) had recurrence of pain. The differences in balloon volume, Meckel's cave size, and compression coefficient were compared among the three groups, and the correlation between balloon volume and Meckel's cave size in each group was analyzed by Pearson correlation. Results: The effective rate of PMC for trigeminal neuralgia was 93.1% (67/72). At time points from T0 to T4, patients had BNI-P scores [M (Q1, Q3)] of 4.5 (4.0, 5.0), 1.0 (1.0, 1.0), 1.0 (1.0, 1.0), 1.0 (1.0, 1.0) and 1.0 (1.0, 1.0), and BNI-N scores [M (Q1, Q3)] of 1.0 (1.0, 1.0), 4.0 (3.0, 4.0), 3.0 (3.0, 4.0), 3.0 (2.0, 4.0) and 2.0 (2.0, 3.0), respectively. Compared with those at T0, patients had lower BNI-P scores and higher BNI-N scores from T1 to T4 (all P<0.05). In all patients, group A, group B, and group C, the balloon volume was (0.65±0.15), (0.67±0.15), (0.59±0.15) and (0.67±0.17) cm3, respectively, with no statistically significant difference (P>0.05), while the Meckel's cave size was (0.42±0.12), (0.44±0.11), (0.32±0.07), and (0.57±0.11) cm3, with a statistically significant difference (P<0.001). The balloon volumes and Meckel's cave sizes were all linearly and positively correlated (r=0.852, 0.924, 0.937 and 0.969, all P<0.05). The compression coefficient in group A, B and C was (1.54±0.14), (1.84±0.18) and (1.18±0.10), respectively, with a statistically significant difference (P<0.001). There were no serious intraoperative complications such as death, diplopia, arteriovenous fistula, cerebrospinal fluid leak, and subarachnoid hemorrhage. Conclusions: Intraoperative balloon volume during PMC for trigeminal neuralgia is linearly and positively correlated with the volume of the patient's Meckel's cave. The compression coefficient varies among patients with different prognoses and the compression coefficient may be a factor affecting the patient's prognosis.

[Medium and long-term follow-up of the Pul-Stent in treating postoperative branch pulmonary artery stenosis in children with congenital heart disease].

Objective: To evaluate the effectiveness and safety of Pul-Stent as the treatment of postoperative branch pulmonary artery stenosis in children with congenital heart disease. Methods: This was a retrospective study. Thirty-three patients who underwent Pul-Stent implantation in Shanghai Children's Medical Center due to postoperative residual pulmonary artery stenosis from August 2014 to June 2015 were included. The immediate curative effect, follow-up and complications of Pul-Stent implantation were assessed. Comparisons between groups were performed with unpaired Student t test. Results: Pul-Stent implantation of 33 patients (19 males and 14 females) were performed successfully. Thirty-one patients underwent percutaneous stenting, and 2 patients underwent hybrid stenting. A total of 35 Pul-Stents were implanted (19 of model small, 15 of model medium and one of model large), 23 stents were planted in the proximal left pulmonary artery and 12 stents were in the proximal right pulmonary artery. The initial diameter of dilation balloon ranged from 6 to 16 mm, and the long sheath of percutaneous implantation ranged from 8 to 10 F in 29 patients (29/31, 94%). After stenting, the diameter of the narrowest segment of pulmonary artery increased from (4.0±1.7) mm to (9.1±2.1) mm in all patients (t=-21.60, P<0.001). The pressure gradient at the stenosis in 26 patients after biventricular correction decreased from (30.5±12.3) mmHg (1 mmHg=0.133 kPa) to (9.9±9.6) mmHg (t=12.92, P<0.001), and the right ventricular to aortic pressure ratio decreased from 0.57±0.14 to 0.44±0.12 (t=7.44, P<0.001). The pressure of the superior vena cava after stenting in 5 patients after cavopulmonary anastomosis decreased from (17.0±1.9) mmHg to (14.0±0.7) mmHg (t=2.86, P=0.046). Two patients died during reoperation for repairing other cardiac malformations. The remaining 31 patients were clinically stable during the follow-up period of (5.3±1.6) years, and one stent fracture was found on chest X-ray. Cardiac catheterization reexaminations in 16 patients showed that restenosis was found in one stent, while stent position and patency were satisfactory in the remaining stents. Nine children underwent post-dilation without stent fracture, displacement or aneurysm formation. Cardiac tomography showed no stent stenosis, fracture observed, or significant change in diameter of the stent in 8 patients. The inner diameter and pulmonary blood perfusion could not be accurately evaluated due to artifacts by cardiac magnetic resonance imaging in 4 patients. Conclusions: Pul-Stent has good compliance and adequate radial strength, and can dilate further over time to accommodate for somatic growth. It performs safely and effectively in treating post-operative branch pulmonary artery stenosis in children.

Serum and exosomal hsa_circ_0000907 and hsa_circ_0057362 as novel biomarkers in the early diagnosis of diabetic foot ulcer.

OBJECTIVE: Diabetic foot ulcer (DFU) is a serious chronic complication leading to disability and death in patients suffering from diabetes. Currently, there is no effective marker for its early diagnosis. The aim of this study is to analyze the difference of circRNA expression profiles between DFU and normal human wounds (NHW) and to screen serum biomarkers for the early diagnosis of DFU. MATERIALS AND METHODS: Differentially expressed circRNAs were screened by bioinformatics analysis, using GSE114248 chip data downloaded from GEO database, including 5 pairs of tissue samples from DFU patients and NHW cases. Accordingly, 20 cases of DFU (Wagner grade 0~2), 20 non-DFU diabetes and 20 healthy controls were selected in the screening test, and the total RNAs of serum and serum-derived exosomes were extracted. The screened circRNAs were verified in the third largest cohort, and the ROC curves were drawn to assess the diagnostic efficiency. RESULTS: As discovered by experiment, there were a total of 67 circRNAs presented differential expressions between the two groups, with 28 circRNAs upregulated and 39 circRNAs downregulated in DFU group. Two types of circRNAs, hsa_circ_0000907 and hsa_circ_0057362, were selected as candidate biomarkers in current study and validated in a large cohort. The AUCs of serum hsa_circ_0000907 and hsa_circ_0057362 to diagnose early DFU were 0.9389 and 0.8792, respectively, and the AUCs of exosomal hsa_circ_0000907 and hsa_circ_0057362 to diagnose early DFU were 0.8783 and 0.8481, respectively. Furthermore, the expressions of serum hsa_circ_0000907 and hsa_circ_0057362 were negatively correlated with ankle brachial index (ABI) and transcutaneous oxygen pressure (TcPO2) in DFU patients. CONCLUSIONS: Serum and exosomal hsa_circ_0000907 and hsa_circ_0057362, especially hsa_circ_0000907, have novel diagnostic capabilities in the early diagnosis of DFU.

3-phenyllactic acid production by free-whole-cells of Lactobacillus crustorum in batch and continuous fermentation systems.

AIM: 3-Phenyllactic acid (3-PLA) has been widely used in food and material industries. Three Lactobacillus crustorum strains have shown greater 3-PLA production ability in our previous study. The objectives of this study were to further improve 3-PLA yields in batch and continuous fermentation systems using of free-whole-cells of the three L. crustorum strains. MATERIALS AND RESULTS: The fermentation conditions of free-whole-cells of the three L. crustorum strains for 3-PLA production were optimized. Among these strains, L. crustorum NWAFU 1078 showed excellent reusability and significantly (P < 0·05) greater 3-PLA production ability than the other strains after 10th recycle. The strain possesses three l-lactate dehydrogenase and three d-lactate dehydrogenase catalysing 3-PLA production from phenylpyruvic acid (PPA). Under the optimal conditions, the strain produced 15·2 mmol l-1 3-PLA (76% PPA conversion rate) in a batch fermentation system and 6·5 mmol l-1  h-1 3-PLA (55% PPA conversion rate) in a continuous fermentation system using a 0·6 dilution rate. CONCLUSIONS: Free-whole-cells of L. crustorum NWAFU 1078 showed excellent reusability and higher 3-PLA yields under optimal biotransformation conditions in both batch and continuous fermentation systems. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the possibility to use the free-whole-cells of L. crustorum NWAFU 1078 as a biocatalyst for effective production of 3-PLA.

[Clinical characteristics of anomalous origin of the left coronary artery from the pulmonary artery in 91 children].

Objective: To explore the clinical manifestations, diagnosis, treatment and prognosis of anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) . Methods: A retrospective study identified 91 patients diagnosed with ALCAPA at Shanghai Children's Medical Center from March 2010 to August 2017. According to the left ventricular ejection fraction (LVEF) at the time of consultation, patients were divided into the cardiac insufficiency group (n=54) and the normal cardiac function group (n=37). Clinical features (age of onset, clinical performance, etc) and auxiliary examinations (electrocardiogram, echocardiography, etc) between the two groups were compared using a t-test and a Chi-square test. Prognostic factors were analyzed by an ordered logistic regression and a Pearson correlation coefficient. Results: (1) The age of diagnosis of patients in the cardiac insufficiency group who were usually misdiagnosed as cardiomyopathy was (10.0±2.6) months (20/54) , whereas the age of diagnosis of patients in the normal cardiac function group who were usually misdiagnosed as valvular diseases was (40.0±7.8) months (4/37). According to the pathophysiological mechanism, forty of the 54 (74%) patients in the cardiac insufficiency group were infantile type, and 78% patients (29/37) in the normal cardiac function group were adult type. (2) Preoperative electrocardiogram showed the deep Q wave in lead I occurred more frequently in the cardiac insufficiency group than in the normal cardiac function group (28/54 vs. 11/37, χ(2)=4.388, P=0.036). (3) Twenty patients died in the cardiac insufficiency group including 12 patients who died from postoperative cardiac pump failure and 8 children who did not undergo surgery due to poor prognosis and died from other reasons. There was no death in the normal cardiac function group. (4) Preoperative LVEF was the unique risk factor affecting prognosis (F=16.872, P=0.005). The preoperative LVEF was significantly lower than the postoperative LVEF ((37±11)% vs. (45±14)%, t=3.614, P=0.001) in the cardiac insufficiency group. During the follow-up period, 6 patients in the cardiac insufficiency group still presented with postoperative cardiac dysfunction, and the patients in the normal cardiac function group still had normal cardiac function. Conclusions: Preoperative LVEF was the unique risk factor affecting prognosis of ALCAPA. Patients with infantile type ALCAPA and preoperative cardiac insufficiency should receive long-term follow-up treatment.

[Clinical analysis of transcatheter closure of ventricular septal defect with right coronary cusp bulge].

Objective: To access the feasibility, effectiveness and safety of transcatheter closure of ventricular septal defect (VSD) with right coronary cusp bulge. Methods: The study population consisted of 40 children (22 boys and 18 girls) undergoing transcatheter intervention for VSD with right coronary cusp bulge in Shanghai Children's Medical Center from August 2013 to June 2017. All patients were examined using transthoracic echocardiography and cardiovascular angiography before occlusion and received transcatheter closure of VSD by placing an occlude. During the operation, angiography and transthoracic echocardiography were used to detect residual shunts, new-onset or aggravation of aortic regurgitation, and surface electrocardiogram was used to assess the impact of occlusion on the conduction system. The children were followed up to evaluate the degree of aortic regurgitation and the presence of heart block by transthoracic echocardiography and electrocardiogram in outpatient department. Comparative analysis between two groups was used by t test. Results: The average age of the patients was (5.1±3.2) years, weight(20.6±10.7) kg. The diameter of defect shunt was (3.3±0.9) cm. The VSD of all 40 patients were successfully occluded immediately, and three patients (8%) with filament residual shunt were observed during the operation. No major surgical complication such as death, pericardial tamponade, embolism of important organs, infective endocarditis occurred during the perioperative period. During the follow-up period, the positions of all the occluders were good, the residual shunt in three patients disappeared, and no new-onset or aggravated aortic regurgitation occurred. Electrocardiogram did not reveal any atrioventricular block, and one patient suffered from an incomplete right bundle branch block. There were significant differences between pre- and post-occlusion regarding the left ventricular end diastolic diameter ((3.8±0.5) vs. (3.7±0.5) cm, t=2.092, P=0.043), the left ventricular end systolic diameter ( (2.4±0.3) vs. (2.2±0.4) cm, t=2.068, P=0.045), and the QRS interval ((75.4±10.4) vs. (79.8±11.4) ms, t=-2.277, P=0.028). No significant differences were found between pre- and post-operation regarding the left ventricular ejection fraction (67.8%±4.7% vs. 68.1%±4.6%, t=-0.447, P=0.657) and the PR interval ((125.6±14.7) vs. (122.6±14.2) ms, t=1.540, P=0.132). Conclusions: Interventional closure of VSD with right coronary cusp bulge has small impact on aortic valve activity in children. With appropriate indications and methods, transcatheter interventional occlusion should be considered because it is effective for children diagnosed with VSD combined with right coronary cusp bulge.

[Radiofrequency catheter ablation of premature ventricular contractions in children under CARTO3 system: a retrospective study from one single center].

Objective: To evaluate the clinical effect and summarize the experience of radiofrequency catheter ablation (RFCA) for children suffered from premature ventricular contractions (PVCs). Methods: This retrospective study was conducted by descriptive analysis. A total of 108 cases with frequent PVCs from Shanghai Children's Medical Center were treated with RFCA under the guidance of CARTO3 system from January 2011 to December 2016. The immediate success rate of the procedure, the recurrence rate and the perioperative complications were summarized. The constituent ratio of different PVCs origins, the trend of overall procedure time and success rate in recent years were analyzed. Statistical analyses were performed using F test. Results: Immediate success of RFCA was achieved in 104 cases (96.3%, 104/108) and 4 cases (3.7%, 4/108) failed. The PVCs recurred during follow-up of over 6 months in 5 cases (4.8%, 5/104) . There were no severe complications related to the procedure. The sites of PVCs origin, in 52 cases originated from right ventricular outflow track (48.2%, 52/108) , 17 cases originated from left ventricular outflow track (15.7%, 17/108) and 26 cases originated from tricuspid annulus (24.1%, 26/108) . Among the three predilection sites of PVCs, the operation time was (141±46) min for right ventricular outflow track, (155±50) min for left ventricular outflow track, and (166±57) min for tricuspid annulus. However, the difference was not statistically significant (F=1.79, P=0.17) . X-ray exposure time was (14±8) minutes for right ventricular outflow track ablation, (32±14) minutes for left ventricular outflow track ablation and (16±8) minutes for tricuspid annulus ablation respectively. The exposure time for the ablation on left ventricle was significantly longer than the other two sites (F=5.12, P=0.018) . Conclusion: RFCA is safe and effective for PVCs in children with high success rate and low recurrence and complication rates.

In Vitro and In Vivo Antibacterial Activities of Patchouli Alcohol, a Naturally Occurring Tricyclic Sesquiterpene, against Helicobacter pylori Infection.

This study further evaluated the in vitro and in vivo anti-Helicobacter pylori activities and potential underlying mechanism of patchouli alcohol (PA), a tricyclic sesquiterpene. In the in vitro assay, the capacities of PA to inhibit and kill H. pylori were tested on three standard strains at different pH values and on 12 clinical isolates. The effects of PA on H. pylori adhesion (and its alpA, alpB, and babA genes), motility (and its flaA and flaB genes), ultrastructure, and flagellation were investigated. Moreover, the H. pylori resistance to and postantibiotic effect (PAE) of PA were determined. Furthermore, the in vivo effects of PA on H. pylori eradication and gastritis were examined. Results showed that MICs of PA against three standard strains (pH 5.3 to 9) and 12 clinical isolates were 25 to 75 and 12.5 to 50 µg/ml, respectively. The killing kinetics of PA were time and concentration dependent, and its minimal bactericidal concentrations (MBCs) were 25 to 75 µg/ml. In addition, H. pylori adhesion, motility, ultrastructure, and flagellation were significantly suppressed. PA also remarkably inhibited the expression of adhesion genes (alpA and alpB) and motility genes (flaA and flaB). Furthermore, PA treatment caused a longer PAE and less bacterial resistance than clarithromycin and metronidazole. The in vivo study showed that PA can effectively eradicate H. pylori, inhibit gastritis, and suppress the expression of inflammatory mediators (COX-2, interleukin 1ß, tumor necrosis factor alpha, and inducible nitric oxide synthase [iNOS]). In conclusion, PA can efficiently kill H. pylori, interfere with its infection process, and attenuate gastritis with less bacterial resistance, making it a potential candidate for new drug development.

Expression of RASSF1A in epithelial ovarian cancers.

OBJECTIVE: Ovarian cancer is the third most common cancer in female reproductive system. But ovarian cancer is hard to detect at early phase. It is very urgent to develop effective early diagnosis method for ovarian cancer. RASSF1A (Ras association domain family 1 isoform A) is a tumor suppressor, which modulates multiple apoptotic and cell cycle checkpoint pathways. We amed to find out the relationship between RASSF1A and ovarian cancer. METHODS: We compared the expressions of RASSF1A gene in different ovarian cancer cell lines, and also in epithelial ovarian cancer tissues and normal ovarian tissues through RT-PCR (reverse transcription polymerase chain reaction) technique. RESULTS: RASSF1A was expressed in HO8910 and HO8910PM cells, while RASSF1A mRNA was absent in SKOV-3 and OVCAR-3 cells. RASSF1A was expressed in 10 normal ovarian tissue samples (10/10, 100%), while RASSF1A was only expressed in 2 ovarian cancers tissue samples (2/47, 4.3%). The difference in the frequency was significant in tissue samples (100% vs. 4.3%, p < 0.05). CONCLUSIONS: RASSF1A could be served as a potential molecular marker for diagnosing ovarian cancer at early phase.

Role of hepcidin in murine brain iron metabolism.

Brain iron homeostasis is maintained by a balance of both iron uptake and release, and accumulating evidence has revealed that brain iron concentrations increase with aging. Hepcidin, an iron regulatory hormone produced by hepatocytes in response to inflammatory stimuli, iron, and hypoxia, has been shown to be the long-sought hormone responsible for the regulation of body iron balance and recycling in mammals. In this study, we report that hepcidin is widely expressed in the murine brain. In cerebral cortex, hippocampus and striatum, hepcidin mRNA levels increased with aging. Injection of hepcidin into the lateral cerebral ventricle resulted in decreased Fpn1 protein levels in cerebral cortex, hippocampus, and striatum. Additionally, treatment of primary cultured neurons with hepcidin caused decreased neuronal iron release and Fpn1 protein levels. Together, our data provide further evidence that hepcidin may be involved in the regulation of brain iron metabolism.

Effects of dietary zinc levels on the activities of enzymes, weights of organs, and the concentrations of zinc and copper in growing rats.

Zinc (Zn) is an essential nutrient that is required in humans and animals for many physiological functions, including immune and antioxidant function, growth, and reproduction. The present study was performed to investigate the effects of three Zn levels, including Zn adequate (35.94 mg/kg, as a control), Zn deficiency (3.15 mg/kg), and Zn overload (347.50 mg/kg) in growing male rats for 6 wk. This allowed for evaluation of the effects that these Zn levels might have on body weight, organ weight, enzymes activities, and tissues concentrations of Zn and Cu. The results showed that Zn deficiency has negative effects on growth, organ weight, and biological parameters such as alkaline phosphatase (ALP) and Cu-Zn superoxide dismutase (Cu-Zn SOD) activities, whereas Zn overload played an effective role in promoting growth, improving the developments of organs and enhancing immune system. Hepatic metallothionein (MT) concentration showed an identical increase tendency in rats fed both Zn-deficient and Zn-overload diets. The actual mechanism of reduction of Cu concentration of jejunum in rats fed a Zn-overload diet might involve the modulation or inhibition of a Cu transporter protein by Zn and not by the induction of MT.

Human ARX gene: genomic characterization and expression.

Arx is a homeobox-containing gene with a high degree of sequence similarity between mouse and zebrafish. Arx is expressed in the forebrain and floor plate of the developing central nervous systems of these vertebrates and in the presumptive cortex of fetal mice. Our goal was to identify genes in Xp22.1-p21.3 involved in human neuronal development. Our in silico search for candidate genes noted that annotation of a human Xp22 PAC (RPCI1-258N20) sequence (GenBank Accession No. AC002504) identified putative exons consistent with an Arx homologue in Xp22. Northern blot analysis showed that a 3.3kb human ARX transcript was expressed at high levels in fetal brain. A 5.9kb transcript was expressed in adult heart, skeletal muscle, and liver with very faint expression in other adult tissues, including brain. In situ hybridization of ARX in human fetal brain sections at various developmental stages showed the highest expression in neuronal precursors in the germinal matrix of the ganglionic eminence and in the ventricular zone of the telencephalon. Expression was also observed in the hippocampus, cingulate, subventricular zone, cortical plate, caudate nucleus, and putamen. The expression pattern suggests that ARX is involved in the differentiation and maintenance of specific neuronal cell types in the human central nervous system. We also mapped the murine Arx gene to the mouse genome using a mouse/hamster radiation hybrid panel and showed that Arx and ARX are orthologues. Therefore, investigations in model vertebrates may provide insight into the role of ARX in development. The recent identification of ARX mutations in patients with various forms of mental retardation make such studies in model organisms even more compelling.

[Ecological risk assessment and its application].

Ecological risk assessment, closely linked to environmental toxicology, is becoming one of the most widely expanding disciplines in environmental science. A number of frameworks for ecological risk assessment have been developed by the USEPA, Environment Canada and European Commission in recent years. This review described the common elements, the process and steps of ecological risk assessment. In addition to drawing upon the recent published literature, we have developed some examples of applications based on our collective experiences in conducting ecological risk assessments.

Evaluation of benomyl and carbendazim in the in vivo aneuploidy/micronucleus assay in BDF1 mouse bone marrow.

Benomyl and its active metabolite carbendazim were investigated in BDF1 mouse bone marrow to establish whether micronuclei induced by these fungicides are caused by clastogenic or aneugenic events. Micronuclei were evaluated for kinetochores using immunofluorescent antikinetochore antibodies. Kinetochore positive (K+) micronuclei are likely to arise from chromosome loss since they presumably contain intact kinetochores and are indicative of aneuploidy. Conversely, kinetochore negative (K-) micronuclei are mostly likely to contain acentric chromosome fragments arising primarily from clastogenic damage. Benomyl and carbendazim were administered as single oral doses of 0.3, 8.6 or 17.2 mmol/kg (for benomyl, equivalent to 100, 2500 or 5000 mg/kg; for carbendazim, equivalent to 66, 1646 or 3293 mg/kg). Both compounds were positive in the micronucleus test at doses of 8.6 and 17.2 mmol/kg, and an average of 82% (benomyl) and 87% (carbendazim) of the total micronucleated polychromatic erythrocytes were K+. No effects were seen with either fungicide at 0.3 mmol/kg. These results are analogous to findings with known aneugens such as vincristine but are in contrast to results with classical clastogens such as cyclophosphamide. Thus, benomyl and carbendazim induce micronuclei in mouse bone marrow cells primarily through an aneugenic mechanism.

Application of the Hydra attenuata assay for identifying developmental hazards among natural waters and wastewaters.

This study concerns application of the Hydra attenuata assay to detect the developmental toxicity potential of various aqueous samples. First, the assay was modified for testing aqueous samples because water quality has a major impact on aquatic toxicity testing and the results thus obtained. Ranges of sample pH, salinity (conductivity), and hardness were examined for their adverse effects upon the hydra. Adult hydra were unaffected morphologically by pH 5.5-9.5, and the artificial embryo ("embryo") developed normally in a pH range of 6.25 to 8.25. For water hardness, the minimal affective concentration was 1000 mg/liter (as CaCO3) in adults and 625 mg/liter in the embryos; the NOAELs for these were 750 mg/liter in the adult and 250 mg/liter CaCO3 in the embryo. Salinity in excess of 5 ppt was lethal to adults and embryos, indicating the assay may not be applicable to marine or highly saline samples. Finally, grab samples were tested from rivers in Maryland, Pennsylvania, New Jersey, and Delaware, some of which are impacted by industrial and agricultural activities, as well as several samples of industrial wastewaters from one major facility. The assay functioned normally with these diverse samples and yielded results that can be used in assessing the potential developmental hazard of these materials.

The in vitro activation of cyclophosphamide in the Hydra Developmental Toxicology Assay.

The proteratogen cyclophosphamide (CP) was tested in the Hydra Assay in the presence and absence of an in vitro metabolic activation package (MAP) consisting of rat hepatic microsomes (0.06 nmol P450/ml), 500 microM NADPH, and 25 microM MgCl. This metabolic system was developed through a series of interrelated biochemical and biological assays to provide maximum cytochrome P450 mixed-function monooxygenase (MFO) metabolic capacity while controlling the inherent toxicity of the hepatic preparation and the attendant cofactors. Bioactivation of CP was confirmed under standard hydra assay conditions of pH 7.0 and 20 degrees C and compared with activation at 37 degrees C. Estimation of total metabolic capacity and verification of activation were made through the appearance of alkylating metabolites both in the absence and in the presence of hydra. Chemical exposure was maintained throughout the 92 +/- 2 hr assay with periodic renewal of media (and additives) at 4, 20, 28, 42, and 66 hr of incubation. Inclusion of bioactivation increased the toxicity of CP by two orders of magnitude. The minimal affective concentration in the adult and developmental portions of the assay was decreased from 4000 to 20 micrograms CP/ml and from 1000 to 4 micrograms CP/ml, respectively. By limiting the inherent toxicity of the MFO package, it was possible to avoid pulse-type exposures and ensure that all ontogenic stages were exposed to active metabolites. The addition of metabolic activation capacity to an in vitro assay, while not essential, markedly enhances its utility and breadth of application in developmental toxicity safety evaluations.

Developmental incompatibility between cell nucleus and cytoplasm as revealed by nuclear transplantation experiments in teleost of different families and orders.

Teleosts from different families and orders were used as materials for nuclear transplantation experiments. (1) The nuclei of goldfish (Carassius auratus, family Cyprinidae, order Cypriniformes) were transplanted into the enucleated egg cytoplasm of loach (Paramisgurnus dabryanus, family Cobitidae, order Cypriniformes) and vice-versa. (2) The nuclei of Tilapia (oreochromis nilotica, order Perciformes) were transplanted into the enucleated egg cytoplasm of goldfish (Carassius auratus, order Cypriniformes). The chromosome number of the nucleus donor fish is different from that of the cytoplasmic recipient fish in each of the two combinations. In the first case, only a few early nucleo-cytoplasmic hybrid (NCH) larval fish were obtained in each combination. In second case, even though a high percentage of NCH blastulas were also obtained, the majority of them died at the same developmental stage, except a few which survived until early gastrula stage. The examination of the metaphase chromosome figures of the NCH blastulas or embryos obtained in all three combinations indicated that they were of nucleus-donor type. The developmental rates of all the NCH eggs were similar to those of cytoplasmic-recipient type. Scanning electronmicroscopy examination showed that the morphology of NCH blastula cells, which were obtained from the combination of Tilapia nucleus and goldfish cytoplasm, manifested obviously abnormal features and the cells were arrested at different stages of cell disintegration. Two-dimension polyacrylamide gel electrophoretograms of the homogenates of Tilapia, goldfish and their NCH blastula cells showed that the protein synthetic pattern of NCH blastula was similar to that of Tilapia nucleus type. The results of experiments which failed to obtain NCH adult fish in all three combinations can be explained as a result of developmental incompatibility between the donor nucleus and the enucleated recipient egg cytoplasm, which were from distantly related fish species. And the chromosome numbers of all the component fish of the three combinations which were examined in the experiment and shown to be quite different from each other in the tested fish, should not be overlooked as one of the essential factors causing the developmental incompatibility in NCH fish in this experiment.