A permanent cell line of the crayfish Orconectes limosus as a potential model in comparative oncology

A cell line, designated OLGA-PH-J/92, was established from neuronal tissue of the crayfish Orconectes limosus. To date the cell line has been subcultured more than 150 times. From the original cell line two 'daughter' cell lines and one cloned cell line were isolated. Best growth was obtained when the cells were incubated in Eagle's Minimum Essential Medium supplemented with 6% fetal bovine serum at a temperature of 27 degrees C. Under these conditions the population doubling time lasted between 23 and 25 hours. The shape of the cells is dendritic, but can change to spherical when conditions are less optimal. The cell lines showed features of transformation, such as anchorage independence, loss of contact inhibition, and low serum requirement. The number of chromosomes found in the cell lines ranged from 11 to 136, while in the donor species numbered between 98 and 106. It will be of interest to study if the O. limosus cell lines grow malignantly in vivo. OLGA-PH-J/92 and the derivative cell lines should also be suitable for studying viral infections in crustaceans.     

Kinetic analysis of uranium accumulation in the bivalve Corbicula fluminea: effect of pH and direct exposure levels

The bioaccumulation of natural uranium in the freshwater bivalve Corbicula fluminea was investigated subsequent to the bivalve's experimental waterborne exposures. A first experiment determined the accumulation rate (transfer efficiency, tissular distribution) and subcellular distribution of uranium in organs after over 42 days of uranium exposure (100 microg l(-1); pH 7) and later following 60 days of depuration. Results showed that there was direct transfer of uranium to the bivalve organs ([U]organism/[U]water = 0.16, fresh weight, fw). The highest accumulation levels occurred in the visceral mass and remained constant throughout the exposure duration, although a linear increase in the U concentration in the gills was observed (2.98 +/- 1.3-10.9 +/- 3.7 microg g(-1) between days 2 and 42). A second set of experiments were performed in order to test the influence of the exposure levels (100; 500; 1500 microg l(-1)) and pH (7 and 8.1) on the bioaccumulation capacities. A marked difference of U distribution is observed as a function of exposure levels (gills were favoured in the case of high exposure levels-relative burden: 49.1 +/- 3% (1500 microg l(-1)), whereas the visceral mass presented higher accumulation levels at environmentally relevant U concentrations). Uranium concentration in the insoluble fraction (80%) in the whole body does not depend upon exposure levels in the water column or upon duration. These experiments did not allow any link to be established between the free-metal ion concentration and the bioaccumulation efficiency. Results showed a significant pH effect and indicated a link between the exposure conditions and the distribution of uranium in the bivalve organs.     

Sodium sulfate impacts feeding, specific dynamic action, and growth rate in the freshwater bivalve Corbicula fluminea

Sodium sulfate is a ubiquitous salt that reaches toxic concentrations due to mining and other industrial activities, yet is currently unregulated at the Federal level in the United States. Previous studies have documented reduced growth of clams downstream of sulfate-dominated effluents, altered bioenergetics in filter-feeding invertebrates, and interactions between sulfate and other toxicants. Therefore, the purpose of this study was to determine if sodium sulfate affects the bioenergetics of the filter-feeding, freshwater bivalve, Corbicula fluminea, and the mechanism by which the effects are elicited. In addition to measuring effects on feeding, respiration and growth rates, I evaluated the relative sensitivity of a green algae consumed by clams to determine if top-down or bottom-up effects might be exhibited under field conditions. This study demonstrated that sodium sulfate had no effect on basal metabolic rates, but significantly reduced the feeding, post-feeding metabolic, and growth rates of C. fluminea. The proposed mechanism for these impacts is that filtering rates are reduced upon exposure, resulting in reduced food consumption and therefore, preventing increased metabolic rates normally associated with post-feeding specific dynamic action (SDA). In the field, these effects may cause changes in whole stream respiration rates and organic matter dynamics, as well as alter uptake rates of other food-associated contaminants like selenium, the toxicity of which is known to be antagonized by sulfate, in filter-feeding bivalves.     

Cytotoxicity assessment of antibiofouling compounds and by-products in marine bivalve cell cultures.

Short-term primary cell cultures were derived from adult marine bivalve tissues: the heart of oyster Crassostrea gigas and the gill of clam Ruditapes decussatus. These cultures were used as experimental in vitro models to assess the acute cytotoxicity of an organic molluscicide, Mexel-432, used in antibiofouling treatments in industrial cooling water systems. A microplate cell viability assay, based on the enzymatic reduction of tetrazolium dye (MTT) in living bivalve cells, was adapted to test the cytotoxicity of this compound: in both in vitro models, toxicity thresholds of Mexel-432 were compared to those determined in vivo with classic acute toxicity tests. The clam gill cell model was also used to assess the cytotoxicity of by-products of chlorination, a major strategy of biofouling control in the marine environment. The applications and limits of these new in vitro models for monitoring aquatic pollutants were discussed, in reference with the standardized Microtox test.     

The key role of metallothioneins in the bivalve Corbicula fluminea during the depuration phase,after in situ exposure to Cd and Zn

An experimental study of the role of metallothioneins (MTs) in Cd and Zn depuration processes in the freshwater bivalve Corbicula fluminea was conducted after in situ exposure on the river Lot (France). Specimens of adult C. fluminea were first transplanted from a lacustrine reference site to a polymetallic polluted station (Bouillac, (B)) for a 42-days' exposure period from September to November 1996. They were then depurated after transfer to the laboratory, and were sub-sampled periodically until May 1997. During the first phase, MT concentrations measured with the Mercury-Saturation Assay were induced for a factor of 3.5 compared with time 0, whereas metal uptake showed accumulation factors of 17 and 4 for Cd and Zn, respectively. During the depuration phase, Cd and Zn concentrations decreased by 18 and 70%, respectively, giving estimated biological half-lives of 500 and 40 days. During the same period, MT concentrations decreased by 37% after transfer under unpolluted conditions, especially between 0 and 3 days, suggesting that MTs play a predominant role in Cd depuration. The quantity of Cd sequestered by the MT fraction, after size-exclusion liquid chromatography, represents on average 40% of the total Cd bioaccumulated in the soft body of the molluscs, compared with only 4-9% for total accumulated Zn. This essential metal was principally bound to low molecular weight proteins, which represented 20% of total Zn. Furthermore, it was observed that MTs had a key role in Cd remanence in the bivalves, and it was also reported that other proteins or small peptides were involved in the depuration of Zn.     

Cadmium biodynamics in the oligochaete Lumbriculus variegatus and its implications for trophic transfer

It has become increasingly apparent that diet can be a major source of trace metal bioaccumulation in aquatic organisms. In this study, we examined cadmium uptake, efflux, and subcellular compartmentalization dynamics in the freshwater oligochaete Lumbriculus variegatus. L. variegatus is an important component of freshwater food webs in Europe and North America and is potentially useful as a standard food source for laboratory-based trophic transfer studies. Cadmium accumulation and depuration were each followed for 10 days. Rate constants of uptake (k(u)) and efflux (k(e)) were estimated and subcellular Cd compartmentalization was followed over the course of uptake and efflux. The partitioning of Cd into operationally-defined subcellular compartments was relatively consistent throughout the 20-day experiment, with the majority of Cd accumulating in the cytosol. No major changes in Cd compartmentalization were observed over uptake or depuration, but there appeared to be some exchange between heat-stable and heat-labile cytosolic protein fractions. Cadmium accumulation from solution was strongly affected by ambient calcium concentrations, suggesting competition between Cd and Ca for uptake sites. Finally, we demonstrate the ability to manipulate the whole body calcium content of L. variegatus as a potential tool for examining calcium influences on dietary Cd dynamics. The potential for this species to be an important conduit of Cd to higher trophic levels is discussed, along with its potential as a standardized food source in metal trophic transfer studies.     

Mercury bioaccumulation and trophic transfer in the terrestrial food web of a montane forest

We investigated mercury (Hg) concentrations in a terrestrial food web in high elevation forests in Vermont. Hg concentrations increased from autotrophic organisms to herbivores < detritivores < omnivores < carnivores. Within the carnivores studied, raptors had higher blood Hg concentrations than their songbird prey. The Hg concentration in the blood of the focal study species, Bicknell’s thrush (Catharus bicknelli), varied over the course of the summer in response to a diet shift related to changing availability of arthropod prey. The Bicknell’s thrush food web is more detrital-based (with higher Hg concentrations) in early summer and more foliage-based (with lower Hg concentrations) during late summer. There were significant year effects in different ecosystem compartments indicating a possible connection between atmospheric Hg deposition, detrital-layer Hg concentrations, arthropod Hg concentrations, and passerine blood Hg concentrations.     

Laboratory and clinical studies of cefminox in the pediatric field

The authors have carried out the pharmacokinetic and clinical studies of cefminox (CMNX, MT-141). The results were as follows: CMNX was given by intravenous drip infusion for 1 hour at a dose of 20 mg/kg b.w. to 2 children. The serum levels of CMNX were 103.02 micrograms/ml and 77.73 micrograms/ml at 1 hour after drip infusion, and the levels at 7 hours were 4.39 micrograms/ml and 4.19 micrograms/ml, respectively. The half life times were 1.20 hours and 1.32 hours, respectively. CSF concentrations of CMNX at 1 hour after drip infusion of a dose of 50 mg/kg in 3 patients with aseptic meningitis were 1.68 micrograms/ml (d.i. for 30 minutes), less than or equal to 0.25 micrograms/ml (d.i. for 1 hour) and 0.51 micrograms/ml (d.i. for 1 hour), respectively. CSF/serum ratios were 1.1% and 0.6%. Clinical efficacy was evaluated in 10 cases with purulent tonsillitis (3 cases), pneumonia (3 cases), pyelonephritis (1 case) and enteritis (3 cases). Excellent and good responses were obtained in all cases. Bacteriological response in the form of eradication was noted 8 of 9 cases. No side effects were observed.     

Laboratory and clinical studies of cefmenoxime in the pediatric field

Laboratory and clinical studies were performed on cefmenoxime (CMX), a new cephalosporin antibiotic, and the following results were obtained. 1. Susceptibility of clinically isolated bacteria to CMX and cefotiam (CTM) or cefazolin (CEZ) Antimicrobial activity of CMX was compared with that of CTM and CEZ against S. aureus, S. epidermidis, S. pneumoniae, H. influenzae and E. coli. CEZ and CTM were more active than CMX against S. aureus, S. epidermidis and S. pneumoniae. But CMX was found to be more active by 1-10 tubes than CEZ and CTM against H. influenzae and E. coli. 2. Clinical efficacy. CMX was intravenously administered to 19 patients; 3 with lacunar tonsillitis, 2 with acute bronchitis, 8 with bronchopneumonia, 3 with UTI, 1 with septicemia, 1 with acute panperitonitis, 1 with S.S.S.S. at daily doses of 30-115 mg/kg (64.6 mg/kg on an average) t.i.d. or q.i.d. for 3-17 days (6.1 days on an average). The overall efficacy rate was 94.7%, i.e., efficacy was excellent in 10 cases (52.6%), good in 8 cases (42.1%), and poor in 1 case (5.3%). Bacteriological efficacy was good, i.e. 16 of the 19 strains disappeared. Transient eosinophilia was observed in 1 patient, but no other laboratory abnormality was observed during treatment. The above results suggest that CMX is 1 of the useful antibiotics in treatment of pediatric infections, especially due to Gram negative bacteria.     

Laboratory and clinical studies of flomoxef in the pediatric field

We have carried out laboratory and clinical studies on flomoxef (FMOX, 6315-S). The results were summarized as follows. FMOX was given by 5-minute intravenous administration to 3 children at a single dose of 20 mg/kg. After the intravenous administration, mean serum levels of FMOX were 110.1 +/- 30.95 micrograms/ml at the end of injection, 44.4 +/- 10.55 micrograms/ml at 15 minutes, 11.0 +/- 1.72 micrograms/ml at 1 hour and 0.42 +/- 0.17 microgram/ml at 6 hours. The mean half-life was 1.14 +/- 0.30 hours. The mean urinary excretion rate was 68.8 +/- 17.4% up to 6 hours after the intravenous administration. FMOX was given by 30-minute drip infusion to 2 children at a single dose of 20 mg/kg and to 3 children at a single dose of 40 mg/kg. After the 30-minute drip infusion, mean peak serum levels of FMOX obtained for the 2 dose levels were 45.5 +/- 0.45 micrograms/ml and 87.4 +/- 18.35 micrograms/ml at the end of injection, respectively, and mean half-lives were 0.63 +/- 0.23 hours and 0.70 +/- 0.27 hours, respectively. The mean urinary excretion rate was 53.4 +/- 6.1% up to 6 hours after the 30-minute drip infusion of 40 mg/kg FMOX. Treatment with FMOX was made in 24 cases of pediatric bacterial infections; 5 cases of purulent tonsillitis, 2 cases of bronchopneumonia, 12 cases of pneumonia, and 1 case each of lymphadenitis, pyothorax, purulent meningitis, cellulitis, and abscess. Results obtained were excellent in 15 cases and good in 9 cases. No significant side effect due to the drug was observed in any cases.     

Laboratory and clinical studies of ceftazidime in the pediatric field

The authors have carried out the laboratory and clinical studies of ceftazidime ( CAZ ) and obtained the following results. The antibacterial activities of CAZ against the clinical isolates of S. aureus, E. coli, K. pneumoniae, E. cloacae, E. aerogenes, S. marcescens, C. freundii and P. aeruginosa were measured by the plate dilution method with inoculum size of 10(6) cells/ml. The susceptibility distribution of S. aureus to CAZ ranged from 3.13 to 100 micrograms/ml, and the peak of distribution was 12.5 micrograms/ml. The peak of susceptibility distribution of E. coli, K. pneumoniae and E. cloacae was 0.2 micrograms/ml, and the distribution of E. aerogenes ranged from 0.1 to 100 micrograms/ml and that of S. marcescens, from 0.05 to 3.13 micrograms /ml. The growth of 92% of P. aeruginosa was inhibited at the concentration of 3.13 micrograms/ml or lower. For pharmacokinetic study, CAZ was given in a single dose of 10 mg/kg by intravenous administration for 5 minutes in 1 child and by drip infusion for 30 minutes in 2 children. After intravenous administration of CAZ , the serum level got to the peak of 41.0 micrograms/ml at 15 minutes, and was 1.0 micrograms /ml at 6 hours. Half-life time was 1.30 hours. With drip infusion of CAZ , the mean peak serum level was 52.45 +/- 2.05 micrograms/ml on completion of the infusion, and 1.05 +/- 0.05 micrograms/ml at 6 hours. Half-life time was 1.30 hours. CAZ was effective in 9 cases out of 11 cases with bacterial infection. No side effect was observed except for elevation of serum GOT and GPT in 1 case and eosinophilia in 1 case.     

Laboratory and clinical studies of aztreonam in the pediatric field

The authors have carried out the laboratory and clinical studies of aztreonam (AZT) and obtained the following results. The antibacterial activities of AZT against the clinical isolates of E. coli, K. pneumoniae and P. aeruginosa were measured by the agar dilution method with inoculum size of 10(6) cells/ml. The susceptibility distribution of E. coli to AZT ranged from 0.025 or lower to 1.56 microgram/ml, and the peak of distribution was 0.05 microgram/ml. The peak of susceptibility distribution of K. pneumoniae was 0.025 microgram/ml or lower, and the distribution of P. aeruginosa ranged from 0.1 to 100 micrograms/ml higher and the peak of distribution was 3.13 micrograms/ml. After intravenous bolus injection of 20 mg of AZT in 4 children, the mean peak serum level was 117 +/- 35.1 micrograms/ml at 15 minutes after injection, and half-life time was 1.42 hours. The mean urinary excretion rates was 63.2 +/- 30.6% up to 6 hours after bolus injection of 20 mg/kg of AZT. AZT was given 11 cases with bacterial injection. Daily doses of AZT were from 41.7 to 94.9 mg/kg. Clinical results obtained were excellent and good responses in 8 of 11 cases (72.7%). No side effect was observed.     

Laboratory and clinical studies on cefodizime in the pediatric field

Laboratory and clinical studies on cefodizime (CDZM, THR-221), a newly developed cephem antibiotic, were done. The results obtained are summarized as follows: 1. Absorption and elimination were examined in a total of 5 cases including a case of 10 mg/kg intravenous drip infusion for 30 minutes, 2 cases of 20 mg/kg rapid intravenous injection and 2 cases of 40 mg/kg drip infusion for 30 minutes. Maximum serum levels were attained immediately after drip infusion or rapid injection. Cmax's were 119.2 micrograms/ml for 10 mg/kg, 374.9 micrograms/ml or 255.7 micrograms/ml for 20 mg/kg, and 321.3 micrograms/ml or 431.8 micrograms/ml for 40 mg/kg. These values were determined using an high performance liquid chromatography (HPLC) method. In general, values using the bioassay were higher than those with the HPLC method. T 1/2 (beta)'s were between 1.74 and 1.93 hours using HPLC, and between 1.77 and 2.24 hours using bioassay. Urinary recovery rates were examined in 3 out of 5 cases. Cumulative urinary recovery rates were 57.9-90.6% with HPLC method and 50.4-88.0% with bioassay in a period of 0-8 hours after administration. 2. Clinical efficacy was evaluated in a total of 22 cases including 14 cases of respiratory tract infections, 5 cases of urinary tract infections and 3 cases of cellulitis. Clinical efficacy rate was 95.2%. Bacteriologically, pathogenic organisms were eradicated in 90.0%. As adverse reactions, 1 angular stomatitis, 1 diarrhea and 1 loose stool were noted. Abnormal laboratory test values detected were 1 case of increased GPT and 1 case of increased GOT and GPT.     

Laboratory and clinical studies on cefminox in the pediatric field

Laboratory and clinical studies were performed as follows on cefminox (CMNX, MT-141), a new cephamycin antibiotic. Pharmacokinetics Serum concentrations of CMNX were measured in 4 patients given CMNX for prophylactic purpose during cardiac catheterization. In 2 patients given 20 mg/kg of CMNX by intravenous bolus injection, the average of peak serum concentration was 178.9 micrograms/ml at 15 minutes. The mean urinary recoveries in these 2 cases was 66.9% within 6 hours after injection. In 2 patients given 20 mg/kg of this drug by 1 hour drip infusion, the peak serum concentration was obtained at the time drip was completed, and the average value was 68.3 micrograms/ml. Clinical efficacy CMNX was administrated intravenously to 13 patients in dose of 52.9 approximately 96.0 mg/kg t.i.d. or q.i.d. for 4 approximately 7 days; 3 with tonsillitis, 6 with bronchitis, 1 with bronchopneumonia, 1 with UTI, 1 with lymphadenitis and 1 with enterocolitis. The overall efficacy rate was 92.3%, i.e., efficacy was excellent in 12, and poor in 1. Bacteriological efficacy was excellent, i.e., 3 of 3 strains disappeared. Side effects were observed in 3 cases, i.e., 1 case of eruption, 1 case of diarrhea and 1 case of transient eosinophilia. The above results suggest that CMNX is a useful antibiotic for treating pediatric bacterial infections.     

Laboratory and clinical studies on aztreonam in the pediatric field

Laboratory and clinical studies were performed as follows on aztreonam (AZT), a new monobactam antibiotic. Pharmacokinetics Serum concentrations of AZT were measured in 1 patient given 20 mg/kg by intravenous bolus injection. The peak concentration was 100 micrograms/ml at 15 minutes, and T 1/2 was 1.85 hours. Clinical efficacy AZT was administrated intravenously to 10 patients in doses of 59.2-170.7 mg/kg (average 76.1 mg/kg) t.i.d. for 3-8 days (average 5.3 days); 5 with pneumonia, 1 with bronchitis, 1 with lymphadenitis, 1 with sepsis (suspected) and 2 with urinary tract infections. The overall efficacy rate was 80%, i.e., efficacy was excellent in 5, good in 3, fair in 1 and poor in 1. Bacteriological efficacy was excellent, i.e., 4 of 4 Gram-negative strains disappeared. Any clinical side effects and laboratory abnormalities were not observed. The above results suggest that AZT is a useful antibiotic for treating pediatric bacterial infections, especially due to Gram-negative bacteria.     

Laboratory and clinical studies on norfloxacin in the pediatric field

In bacteriological, pharmacokinetic and clinical studies on norfloxacin (NFLX, AM-715), the following results were obtained: 1. Antibacterial activity of NFLX, nalidixic acid (NA), amoxicillin (AMPC), cefaclor (CCL), erythromycin (EM) and fosfomycin (FOM) against clinically isolated bacteria was examined, and it was found that MIC80 of NFLX against Staphylococcus aureus was 3.13 micrograms/ml, thus NFLX exhibited stronger antimicrobial activity than NA, AMPC, CCL, EM and FOM. NFLX also showed good activities to those strains of S. aureus which were resistance to NA, AMPC, CCL, EM and FOM. 2. MIC80 of NFLX against Escherichia coli was 0.05 micrograms/ml or lower, thus NFLX showed better activity than NA, AMPC, CCL, EM and FOM. 3. In single oral administration at fasting of NFLX at dose levels of 1.5-2.4 and 2.5-3.4 mg/kg in tablet form mean peak values of serum concentration were 0.32 micrograms/ml reached in 1 hour and 0.38 micrograms/ml in 2 hours, T1/2's obtained were 1.7-4.0 and 2.2-2.9 hours and AUC's were 1.54 +/- 0.52 and 2.02 +/- 0.93 micrograms.hr/ml, respectively. Urinary recovery rates of 11.6-46.9%, 13.8-35.4% in 6-8 hours were demonstrated with the 2 ranges of dose levels, respectively. 4. NFLX was administrated to 34 patients consisting of 8 cases of acute pneumonia, 3 cases of acute tonsillitis, 3 cases of bacterial colitis, 19 cases of urinary tract infections and 1 case of purulent parotitis. The clinical efficacy rate was 97.1% including 34 cases with excellent results in 28, good in 5 and fair in 1. 5. The bacterial eradication rate was 96.8% (30/31) with one exception of a Campylobacter jejuni strain. 6. NFLX was given to patients according to a dosing regimen with 4.5-21.4 mg/kg/day dose levels for 3 doses daily except 1 case of UTI where 2 daily doses were given daily. 7. No adverse reactions were observed. Abnormal laboratory test value was obtained in 1 case where eosinophilia was found. The above results have suggested that NFLX is a useful and safe antimicrobial agent against bacterial infections in children.