2004 Vol. 17, No. 3

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Protective Effect of Distillate and Redistillate of Cow's Urine in Human Polymorphonuclear Leukocytes Challenged With Established Genotoxic Chemicals
2004, 17(3): 247-256.
From the ancient period cow's urine has been used as a medicine. In Veda, cow's urine was compared to the nectar. In Susrut, several medicinal properties of cow's urine have been mentioned and are known to cause weight loss, reversal of certain cardiac and kidney problems, indigestion, stomach ache, edema, etc. However, the literature and scripture did not mention the antigenotoxic properties of cow's urine. Methods In the present investigation, the antigenotoxic/ antioxidant properties of cow's urine distillate and redistillate were studied in vitro. The antioxidant status and volatile fatty acid levels were determined. Actinomycin-D (0.1 μmol/L) and hydrogen peroxide (150 μmol/L) were used for inducing DNA strand break with 0.1% DMSO as negative control. Dose for the antigenotoxic effect of cow's urine was chosen from the dose response study carried out earlier. Results Both actinomycin-D and H2O2 caused statistically significant DNA unwinding of 80% & 75% respectively (P<0.001) as revealed by fluorimetric analysis of DNA unwinding (FADU), and the damage could be protected with the redistilled cow's urine distillate (1, 50 & 100 μL) in simultaneous treatment with genotoxic chemicals. Conclusion The redistillate of cow's urine was found to possess total antioxidant status of around 2.6 mmol, contributed mainly by volatile fatty acids (1500 mg/L) as revealed by the GC-MS studies. These fatty acids and other antioxidants might cause the observed protective effects.
Genotoxicity of Pesticide Waste Contaminated Soil and Its Leachate
2004, 17(3): 257-265.
Improper land disposal of hazardous waste can result in leaching of hazardous constituents which may contaminate ground and surface water leading to adverse impact on human health and environment consequences. The present study utilized mammalian cell culture for the genotoxicity assessment of waste and its leachate. Methods Genotoxic potential and chemical analysis of pesticide derived tarry waste contaminated soil extract and its leachate was assessed using in vitro human lymphocyte cultures and GC-MS. Results The investigation revealed that the soil extract could cause significant to highly significant genotoxicity in the form of DNA strand break at 25 μL (P<0.01), 50 μL, 100 μL and 200 μL (P<0.001) and chromosomal aberration at 25 μL (P<0.01) and 50 μL and 100 μL (P<0.001). The leachate could cause significant DNA strand break and chromosomal aberration only at 100 μL and 200 μL (P<0.01) dose levels. Conclusion The genotoxicity observed is attributed to carbaril and tetra methyl naphthyl carbamate, the major ingredients of the extracts, as revealed by GC-MS.
May Chronic Childhood Constipation Cause Oxidative Stress and Potential Free Radical Damage to Children?
2004, 17(3): 266-272.
To investigate whether chronic childhood constipation (CCC) may cause oxidative stress and potential free radical damage to children, and to explore the mechanisms by which CCC may cause oxidative stress and potential free radical damage to chronic constipation patients (CCPs). Methods Sixty CCPs and sixty healthy child volunteers (HCVs) whose ages, gender and others were matched for the CCPs were enrolled in a randomized controlled study, in which levels of vitamin C (VC) and vitamin E (VE) in plasma as well as activities of superoxide dismutase (SOD) and catalase (CAT) in erythrocytes were determined by spectrophotometric analytical methods. Results Compared with average values of the above biochemical parameters in the HCVs group, the average values of VC and VE in plasma as well as those of SOD and CAT in erythrocytes in the CCPs group were significantly decreased (P<0.0001). Linear regression and bivariate correlation analysis showed that with prolonged course of the CCPs, the levels of VC and VE in plasma as well as the activities of SOD and CAT in erythrocytes in the CCPs were decreased gradually (P<0.0001). Conclusion The findings in the present study suggest that chronic childhood constipation causes oxidative stress and potential free radical damage to children with chronic constipation.
Study of a Novel Antiosteoporosis Screening Model Targeted on Cathepsin K
2004, 17(3): 273-280.
To establish an effective assay to access the effects of natural products on cathepsin K for screening antiosteoporosis drugs. Methods To obtain the purified cathepsin K, we cloned the target fragment from the mRNA of human osteosacoma cell line MG63 and demonstrated its correctness through DNA sequencing. Cathepsin K was expressed in a high amount in E.coli after IPTG induction, and was purified to near homogenetity through resolution and column purification. The specificity of the protein was shown by Western blotting experiment. The biological activity of the components in the fermentation broth was assayed by their inhibitory effects on cathepsin K and its analog papain. Results With the inhibition of papain activity as a screen index, the fermentation samples of one thousand strains of fungi were tested and 9 strains among them showed strong inhibitory effects. The crude products of the fermentation broth were tested for their specific inhibitory effects on the purified human cathepsin K, the product of fungi 2358 shows the highest specificity against cathepsin K. Conclusions The compounds isolated from fungi 2358 show the highest biological activity and are worth further structure elucidation and function characterization.
Changes in Synapses and Axons Demonstrated by Synaptophysin Immunohistochemistry Following Spinal Cord Compression Trauma in the Rat and Mouse
2004, 17(3): 281-290.
and methods To evaluate synaptic changes using synaptophysin immunohistochemstry in rat and mouse, which spinal cords were subjected to graded compression trauma at the level of Th8-9. Results Normal animals showed numerous fine dots of synaptophysin immunoreactivity in the gray matter. An increase in synaptophysin immunoreactivity was observed in the neuropil and synapses at the surface of motor neurons of the anterior horns in the Th8-9 segments lost immunoreactivity at 4-hour point after trauma. The immunoreactive synapses reappeared around motor neurons at 9-day point. Unexpected accumulation of synaptophysin immunoreactivity occurred in injured axons of the white matter of the compressed spinal cord. Conclusion Synaptic changes were important components of secondary injuries in spinal cord trauma. Loss of synapses on motor neurons may be one of the factors causing motor dysfunction of hind limbs and formation of new synapses may play an important role in recovery of motor function. Synaptophysin immunohistochemistry is also a good tool for studies of axonal swellings in spinal cord injuries.
Polymorphism of N-acetyltransferase 2 (NAT2) Gene Polymorphism in Shanghai population:Occupational and Non-occupational Bladder Cancer Patient Groups
2004, 17(3): 291-298.
Arylamine N-acetyltransferases (NATs) are involved in the detoxification of aromatic amines and hydrazine. In order to explore the possible association of NAT2 polymorphism with bladder cancer risk in benzidine exposed or non-exposed Chinese individuals, healthy subjects, subjects with bladder cancer of a former benzidine exposed cohort in Shanghai dyestuff industry and a group of bladder cancer patients without known occupational exposure to aromatic amines were genotyped for NAT2 gene polymorphism. Methods NAT2 genotyping was performed with a set of RFLP procedures at seven major polymorphic loci of gene coding area: G191A, C282T, T341C, C481T, G590A, A803G and G857A. Results The wild allele NAT2 *4 was the most prevalent allele (59%) in healthy individuals. The alleles NAT2*6A and NAT2*7B were also frequently observed (21% and 17%, respectively). In contrast to Caucasians, the percentage of slow acetylators was lower (12% in Chinese vs. 58% in Caucasians, P<0.001). No relevant differences were observed for homogenous rapid, heterogeneous rapid/slow and homogeneous slow acetylation genotypes between the healthy subjects and both groups of bladder cancer patients. Conclusion The present work did not support the association of slow acetylating genotypes of NAT2 gene with elevated risk of bladder cancer in Chinese whereas it was documented as an important genetically determined risk factor in Caucasians. Different mechanisms might play a role in individual susceptibility to bladder cancer related with aromatic amine exposure in various races or ethnic groups.
Occurrence of Haloacetic Acids in Drinking Water in Certain Cities of China
HONG Zhou, Xiao-jian ZHANG, Zhan-sheng WANG
2004, 17(3): 299-308.
Since haloacetic acids (HAAs), which are nonvolatile and of high carcinogenic risk, are common species of chlorinated disinfection by-products(DBPs) in drinking water, and little has been known in China, it is necessary to make a survey about the kinds and levels of HAAs in drinking water of the nation. Method HAAs were analyzed using gas chromatography with electron capture detector(GC/ECD) and relatively complex pretreatment process of sample was applied. Five main cities in different areas of China were chosen in the survey. Results Studies showed that the main species of HAAs in drinking water in China were DCAA and TCAA, ranging from 0.4 μg/L to 12.85 μg/L and from 0.56 μg/L to 10.98 μg/L, respectively. MBAA and DBAA were also detected in one city, ranging from 2.20 μg/L to 4.95 μg/L and 1.10 μg/L to 2.81 μg/L, respectively. Therefore, the contents of HAAs varied, usually no more than 25 μg/L. Based on the acquired data to date, it is known that the concentrations of HAAs in drinking water in China were surely under the limits of Sanitary Standard for Drinking Water Quality (China, 2001). Conclusion A wider survey of HAAs in drinking water should be conducted throughout the nation to get adequate data and information, the ultimate aim of which is to control HAAs pollution and keep the balance between microbiological safety insurance and chemical risk control, minimize the formation of DBPs and ensure the safety of water supply at the same time.
Hepatotoxic Alterations Induced by Subchronic Exposure of Rats to Formulated Fenvalerate (20% EC) by Nose Only Inhalation
2004, 17(3): 309-314.
Fenvalerate (20% EC) is a synthetic pyrethroid, which is commonly used in India by farmers for the protection of many food and vegetable crops against a wide variety of insects. However, its inhalation toxicity data is very limited in the literature due to the fact that the exposure levels associated with these effects were usually not reported. Hence, inhalation exposure was carried out to investigate the hepatotoxic effects. Method Adult male rats were exposed to fen for 4 h/day, 5 days a week for 90 days by using Flow Past Nose Only Inhalation Chamber. Sham treated control rats were exposed to compressed air in the inhalation chamber for the same period. Results The results indicated hepatomegaly, increased activities of serum clinical enzymes (indicative of liver damage/dysfunction) along with pronounced histopathological damage of liver. Conclusion The hepatotoxic potential of formulated Fen (20% EC) in rats exposed by nose only inhalation is being reported for the first time and warrant adequate safety measures for human beings exposed to this insecticide, particularly by inhalation route.
Induction of Heme Oxygenase-1 in Human Hepatocytes to Protect Them From Ethanol-induced Cytotoxicity
2004, 17(3): 315-326.
We investigated the relationship between ethanol exposure and heme oxygenase (HO-1) in human hepatocytes in order to ascertain if induction of HO-1 can prevent ethanol induced cellular damage. Methods Dose-dependent (25-100 mmol/L) and time-dependent (0-24 h) ethanol exposure were used in the present study. HO-1 mRNA and protein expression were detected by PT-PCR and Western blot respectively. HO-1 activity was indicated by bilirubin and Fe2+ formation. Cytotoxicity was investigated by means of lactate dehydrogenate (LDH) and aspartate transaminase (AST) level in culture supernatants, as well as the intracellular formation of malondialdehyde (MDA), cellular glutathione (GSH) status and CYP 2E1 activity. Results We first demonstrated a dose-dependent response between ethanol exposure and HO-1 mRNA and protein expression in human hepatocytes. We further observed a time-dependent increase of HO-1 mRNA expression using 100 mmol/L ethanol starting 30 minutes after ethanol exposure, reaching its maximum between 3 h and 9 h. Being similar to what had been demonstrated with the mRNA level, increased protein expression started at 6 h after ethanol exposure, and kept continuous elevated over 18 h. In addition, we found that ethanol exposure to hepatocytes markedly increased HO-1 enzyme activity in a time-dependent manner measured as bilirubin and Fe2+ formation in human hepatocytes. Our results clearly showed that ethanol exposure caused a significant increase of LDH, AST, and MDA levels, while the antioxidant GSH was time-dependently reduced. Furthermore, we demonstrated that pre-administration of cobalt protoporphyrin (CoPP) induced HO-1 in human hepatocytes, and prevented an increase of MDA and a decrease of GSH. These effects could be partially reversed by zinc protoporphyrin (ZnPP), an antagonist of HO-1 induction. Conclusion HO-1 expression in cells or organs could lead to new strategies for better prevention and treatment of ethanol-induced oxidative damage in human liver.
Expression of JunB Induced by X-rays in Mice
2004, 17(3): 327-332.
To explore JunB gene expression in spleen cells of mice after the whole body irradiation as well as in normal hematopoietic and leukemia cells in the primary culture after different dosages of X-ray irradiation. Methods Spleen cells were isolated from the mice irradiated with 3 Gy X-rays. Primary cultured cells from mice were incubated in different intervals after X-irradiation at different dosages. Total RNA was extracted from the cells and the fluctuation of JunB mRNA level was assessed by the RNA ratio of JunB/β-actin measured by quantitative Northern blot hybridization. Results After the mice were exposed to 3 Gy X-rays irradiation, JunB expression in spleen cells was remarkably and rapidly increased, and reached its peak 0.5 h later in C3H/He mice and 1 h later in Balb/c mice. In the primary culture of normal spleen and leukemia cells, JunB mRNA levels increased 30 min after irradiation. The enhanced levels of JunB mRNA were returned to a normal level within 240 min after irradiation. Conclusions JunB gene is responsive to ionizing irradiation and is induced at immediate-early phase after the stimulation. This suggests that the JunB gene plays an important role in the early process of the cells against radiation.
Trends in Injury-related Incidence and Mortality Among Inpatients in Guangdong Province in 1997-2001
2004, 17(3): 333-340.
Currently, China is in short of thorough and systemic data concerning the patterns and incidence of injuries and related deaths. Guangdong Province as one of the economically advanced areas in China is faced with a relatively serious injury problem, and investigation of this problem in this Province will provide valuable information for other provinces and areas in this Country, as well as scientific basis for policy making for injury prevention and control. Methods Analyses are based on the computerized hospital discharge data collected from 322 hospitals in Guangdong Province between 1997 and 2001. Diagnoses are coded according to the International Classification of Diseases, 9th Revision, Clinical Modification (ICD-9-CM). Results The total hospitalization rate related with injuries increased year by year from 1997 to 2001. The ratio of case-fatality has a decline trend for all injury inpatients, who were mainly caused by motor vehicle accidents, unintentional falls, puncture and cut by machine and others. The constituent ratio of death among patients caused by motor vehicle accidents accounted for 56.13% among the total deaths, which ranked as the first place. The direct medical cost also had an increased trend. Conclusions Data on injuries requiring hospitalization can be used to design and target more effective injury prevention programs. Injury prevention would decrease human sufferings, disability, and associated economic losses.
Effect of Dynein Inhibitor on Mouse Oocyte in vitro Maturation and Its Cyclin B1 mRNA Level
2004, 17(3): 341-349.
To evaluate the effect of dynein inhibitor on mouse oocyte in vitro maturation and its cyclin B1 transcription level. Methods Immature mouse oocytes were cultured in vitro with a known dynein ATPase activity inhibitor-sodium orthovanadate (SOV) to detect the changes of maturation rate, and semi-quantitative RT-PCR and single cell RT-PCR were performed to detect the changes of cyclin B1 mRNA level. Results In dose-dependent experiments, the maturation rates of oocytes were significantly different between 5 (mol/L SOV and control groups (P<0.05), and decreased with SOV increasing doses. However, the elevation of cyclin B1 mRNA level of immatured oocytes cultured for 12 h depended on SOV concentrations ranging from 50 to 500 (mol/L. In incon- tinuity exposed SOV experiments, the maturation rates of oocytes markedly reduced after the first incubation with 400 (mol/L SOV at least for 1 h and were first cultured in SOV-free medium for 4 h or 8 h before exposure to SOV (P<0.05). In time-course experiment, the opposite changes of cyclin B1 mRNA level in oocytes between SOV and control groups were observed. Conclusion Dynein inhibitor might delay oocytes meiosis process, and cause ectopic expression of cyclin B1 in oocytes. Most Oocytes incubated with SOV blocked at germinal vesicles (GV) stage or MⅠto anaphase transition due to dynein dysfunction and ectopic transcription level of cyclin B1.
Identification of a New Peptide Deformylase Gene From Enterococcus faecium and Establishment of a New Screening Model Targeted on PDF for Novel Antibiotics
2004, 17(3): 350-358.
To identify a new peptide deformylase (PDF) gene (Genebank Accession AY238515) from Enterococcus faecium and to establish a new screening model targeted on PDF. Methods A new PDF gene was identified by BLAST analysis and PCR and was subsequently over-expressed in the prokaryotic expression host E.coli Bl21(DE3). Over-expressed protein was purified for enzymatic assay by metal affinity chromatography and a new screening model was established for novel antibiotics. Result A new PDF gene of Enterococcus faecium was identified successfully. Ten positive samples were picked up from 8000 compound library and the microbial fermentation broth samples. Conclusion A new PDF of gene Enterococcus faecium was first identified and the model had a high efficacy. Positive samples screened may be antibacterial agents of broad spectrum.
Evaluation of Inhibitory Effect of the Plant Phyllanthus amarus Against Dermatophytic Fungi Microsporum gypseum
2004, 17(3): 359-365.
The antifungal activity of various solvent extracts (such as ether, chloroform, ethyl acetate and ethyl alcohol) of the plant Phyllanthus amarus against dermatophytic fungi Microsporum gypseum was observed. Method Antifungal bioassay in terms of reduction in weight, colony diameter and sporulation of the target fungal colony was carried out using Broth Dilution method. Results Root part of the plant, extracted in various organic solvents did not show any noticeable antifungal activity. The percentage inhibition observed in different solvent extracts of aerial part was found as reduction in weight: chloroform [50.3%], ethyl acetate [27.7%] and ethyl alcohol [12.1%], reduction in colony diameter: chloroform [53.4%], ethyl acetate [31.4%] and ethyl alcohol [15.0%] and reduction in sporulation: maximum inhibition in chloroform extract, at test concentration of 4000 ppm at incubation period of 8 days. Conclusion Chloroform fraction of the aerial part of the plant P. amarus shows significant inhibitory effect against dermatophytic fungi M. gypseum and requires chemical characterization for its bioactive principle.
Statistical Distributions of Ambient Air Pollutants in Shanghai,China
2004, 17(3): 366-372.
To determine the best statistical distribution of concentration data of major air pollutants in Shanghai. Methods Four types of theoretic distributions (lognormal, gamma, Pearson V and extreme value) were chosen to fit daily average concentration data of PM10, SO2 and NO2 from June 1, 2000 to May 31, 2003 in Shanghai by using the maximum likelihood method. The fit results were evaluated by Chi-square test. Results The best-fit distributions for PM10, SO2 and NO2 concentrations in Shanghai were lognormal, Pearson V, and extreme value distributions, respectively. Conclusion The results can be further applied to local air pollution prediction and control, e.g., the probabilities exceeding the air quality standard and emission source reduction of air pollutant concentration to meet the standard.
Wastewater Treatment in a Hybrid Biological Reactor (HBR) :Nitrification Characteristics
2004, 17(3): 373-379.
To investigate the nitrifying characteristics of both suspended- and attached- biomass in a hybrid bioreactor. Methods The hybrid biological reactor was developed by introducing porous ceramic particles into the reactor to provide the surface for biomass attachment. Microorganisms immobilized on the ceramics were observed using scanning electron microscopy (SEM). All chemical analyses were performed in accordance with standard methods. Results The suspended- and attached-biomass had approximately the same nitrification activity. The nitrifying kinetic was independent of the initial biomass concentration, and the attached-biomass had a stronger ability to resist the nitrification inhibitor. Conclusion The attached biomass is superior to suspended-biomass for nitrifying wastewater, especially that containing toxic organic compounds. The hybrid biological reactor consisting of suspended- and attached-biomass is advantageous in such cases.