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Microplates with integrated oxygen sensors for kinetic cell respiration measurement and cytotoxicity testing in primary and secondary cell lines.

This paper presents a cytotoxicity and cell respiration assay that is nondestructive and kinetic. It makes use of 96-well microplates integrated with oxygen sensors. The oxygen signal monitored on-line gives an indication of the cell viability. We show its application for suspension cell lines (Chinese hamster ovary and HL60 cells) as well as adherent (Caco2 cells) and primary (rat hepatocytes) cells using well-known cytotoxic compounds (sodium azide, diclofenac, clozapine, sodium dodecyl sulfate, 2-thiouracil, tamoxifen, and tranylcypromine). The 50% lethality concentration (LC50) obtained from the assay is compared with the standard 3-(4,5-dimethylthiazol-2- yl)-2,5-diphenyl-2H-tetrazolium bromide end-point assay. The cells can be grown directly in the plates, and the assay requires no further reagents or processing. The cells can be harvested for further analysis, if required. The on-line dynamic measurement allows the calculation of LC50 as a function of exposure time. LC50 was shown to decrease with time in HL60 cells. The dynamics of this process was considerably different for the three compounds sodium dodecyl sulfate, tamoxifen, and diclofenac, indicating a large potential of application of this method for cell death studies. The assay system can be applied to almost any cell-based systems with little adaptation. The assay is robust, flexible, and applicable for medium- to high-throughput systems requiring only minimal handling and no additional agent.

Animals↗

Preoperative intravenous fluid therapy decreases postoperative nausea and pain in high risk patients.

The potential for preoperative IV rehydration to reduce postoperative nausea and vomiting (PONV) and pain in patients undergoing ambulatory surgery remains unclear, with conflicting results reported. We sought to determine whether preoperative IV rehydration with a balanced salt solution would decrease the incidence of PONV in patients at increased risk for these symptoms. Eighty ASA grade I-III patients presenting for gynecologic laparoscopy were randomized to receive large (2 mL/kg per hour fasting) or small (3 mL/kg) volume infusions of compound sodium lactate solution over 20 min preoperatively. A standardized balanced anesthetic was used. The incidence and severity of PONV and pain, and need for supplemental antiemetic and analgesic therapy, were assessed by a blinded investigator at 0.5, 1, and 4 h postoperatively, and on the first and third postoperative days. The incidence (control 87% versus large volume 59%) and severity of PONV were significantly reduced in the large volume infusion group at all time intervals. The large volume infusion group also had decreased postoperative pain scores and required less supplemental analgesia. Preoperative correction of intravascular volume deficits effectively reduces PONV and postoperative pain in high risk patients presenting for ambulatory surgery. We recommend the preoperative administration of 2 mL/kg of compound sodium lactate for every hour of fasting to patients with an increased PONV risk presenting for ambulatory surgery.

Adult↗

Interaction of alkylmercuric compounds with sodium selenite. II. Metabolism of methylmercuric chloride administered alone and in combination with sodium selenite in rats.

Repeated doses of sodium selenite (Se) were administered to rats receiving repeated (IV or PO) doses of 0.25 or 2.5 mg Hg/kg methylmercuric chloride (Me2(203)Hg). Se (0.5 mg/kg) was observed to alter the distribution of Me203Hg among tissues as well as among subcellular fractions of kidneys and liver. An excess of selenium resulted in a twofold decrease in the mercury content of kidneys and a similar increase in the mercury content of brain.

Animals↗

The antiviral activity against herpes simplex virus of the triterpenoid compounds carbenoxolone sodium and cicloxolone sodium.

Dose-response experiments show that the presence of 300 microM cicloxolone sodium (CCX) or 500 microM carbenoxolone sodium (CBX) during the HSV replication cycle reduced the infectious virus yield by 10,000- to 100,000-fold: CCX is the more potent anti-herpes agent. HSV-2 replication was consistently more severely restricted by either drug than was that of HSV-1. The ED50 values obtained for either drug against HSV-1 or HSV-2 correlate well with data from dose-response curves. CCX, and to a lesser extent CBX, can be cytotoxic but the degree of cytotoxicity varies between cell lines and is also affected by the physiological state of the cells. Triterpenoid drugs exhibit some activity against virus particles in suspension but the effect is small and contributes little to the overall antiviral effect. The drugs appear to be active throughout the replication cycle. In contrast to all other anti-herpesvirus agents in clinical use the triterpenoid compounds do not appear to act directly to block virus DNA synthesis. HSV mutants resistant to ACG and PAA, or lacking a thymidine kinase gene, appear as sensitive as wild-type virus to CCX inhibition. HSV growth in the presence of the drugs resulted in a lower number of assembled virus particles but reduced to a much greater extent the infectious virus yield: thus the progeny virus quality is greatly diminished. Thermolability of progeny virus correlated well with this diminution of quality in increasing CCX concentration. SDS PAGE analysis of the structural proteins of virus particles made in cells treated with 300 microM CCX revealed numerous differences in the relative intensities of protein bands, which is in keeping with the changed quality of the drug-produced virus. SDS PAGE analysis of the polypeptides induced in drug treated infected cells revealed two effects; some polypeptides were synthesised in reduced amounts and the nuclear/cytoplasmic distribution of certain proteins was affected. Post-translational processing by glycosylation and sulphation of both cellular and HSV induced proteins was strongly inhibited by the triterpenoid drugs, while phosphorylation of only a few polypeptides appeared to be affected.

Animals↗

Genotoxicity of nitroso compounds and sodium dichromate in a model combining organ cultures of human nasal epithelia and the comet assay.

Genotoxic effects of xenobiotics are a possible step in tumor initiation in the mucosa of the upper aerodigestive tract. Using the comet assay, detecting genotoxicity in human tissue has been restricted to single incubations in vitro, but in vivo most xenobiotics harm their target in a repetitive or chronic manner. Therefore, we propose a model, which provides repetitive incubations in human upper aerodigestive tract mucosa cultures. Samples of human inferior nasal turbinate mucosa (n = 25) were cultured according to a modified version of a technique originally described by Steinsvåg. On day 1 fresh samples and on days 7, 9 and 11 organ cultures were incubated with N-nitrosodiethylamine (NDEA), sodium dichromate (Na2Cr2O7) and N'-methyl-N-nitro-N-nitrosoguanidine (MNNG). Mucosa samples and organ cultures, respectively, underwent a modified comet assay on days 1, 7 and 11. Genotoxicity could be shown for NDEA, Na2Cr2O7 and MNNG on days 1, 7 and 11. Duration of tissue culture and repetitive incubations did not significantly influence the results for NDEA. Nevertheless, Na2Cr2O7 and MNNG caused higher genotoxic effects on cultures subjected to the comet assay on day 11. This model may help to assess genotoxic hazards posed by environmental pollutants that have a cumulative character in repetitive or chronic exposure in vivo.

Adult↗