Modulation of brain serotonin by benzyl butyl phthalate in Fundulus heteroclitus (mummichog).

Endocrine-disrupting chemicals have been known to alter important animal behaviors by modulating serotonin (5-hydroxytryptamine, 5-HT) and dopamine. F. heteroclitus (mummichog) brain serotonin and dopamine levels were quantified by enzyme-linked immunosorbent assay (ELISA) following a 28-day exposure regimen involving daily doses of either 0.1 mg l-1 benzyl butyl phthalate (BBP) dissolved in acetone or acetone alone (0.1 mg l-1). No differences in mean brain mass or total protein homogenate were induced by exposure to the acetone vehicle or BBP in acetone. The acetone vehicle had no effect on dopamine, serotonin, or tyrosine hydroxylase levels, but acetone did decrease tryptophan hydroxylase levels (p = 0.011). Exposure to BBP in acetone decreased dopamine (p = 0.024), increased serotonin (p < 0.001), reduced tryptophan hydroxylase as compared to the acetone vehicle alone (p < 0.001), and had no significant effect on tyrosine hydroxylase levels. This study is the first to report modulation of F. heteroclitus brain serotonin and its enzyme tryptophan hydroxylase following sub-lethal exposure to BBP in an acetone vehicle. In addition, modulation of brain dopamine in F. heteroclitus, sans simultaneous modulation of tyrosine hydroxylase, was also observed. These findings support the use of F. heteroclitus for assessing sub-lethal BBP exposure.

Isotopic ((13)C) analysis of dissolved organic carbon in stream water using an elemental analyzer coupled to a stable isotope ratio mass spectrometer.

A technique for measurement of the stable isotope composition of dissolved organic carbon (DOC) in stream water, using an elemental analyzer (EA) coupled to an isotope ratio mass spectrometer (IRMS), is described. Stream water samples were concentrated by rotary evaporation, acidified to remove dissolved inorganic carbon (DIC), and dried in silver cups prior to analysis. Precision was evaluated with standards (alanine and humic acid), and with stream water samples with varying (13)C enrichment. Standards and samples were also prepared in sealed quartz tubes for high-temperature combustion (HTC) and analyzed by dual inlet for comparison. The delta(13)C values of natural abundance standards and samples measured by the two techniques differed by

Autecological properties of 3-chlorobenzoate-degrading bacteria and their population dynamics when introduced into sediments.

Ecologically significant properties of wild-type and genetically engineered bacteria capable of degrading 3-chlorobenzoate (3-CB) were compared in the laboratory, and isolates were introduced into streambed sediments in microcosms to observe their population dynamics. 3-CB metabolism, growth on algal extract, temperature optima, and ingestion by protozoa were ecological properties considered relevant to the persistence of these bacteria if introduced into nature. Cell-specific Vmax for 3-CB metabolism and cell-specific mineralization rates each spanned approximately 2 orders of magnitude, but isolates did not rank consistently. The Ks for 3-CB metabolism for Alcaligenes sp. BR60 was approximately 40-fold lower than the mean value for the other isolates, which differed only approximately 4-fold among themselves. All isolates grew on an algal extract nearly as well as on tryptone-yeast extract, implying potential for survival on natural metabolic substrates in situ. Most isolates had temperature optima that were 3-15 degrees C higher than maximum stream water temperature (22 degrees C). Ciliates preferentially ingested P. acidovorans M3GY, and either P. putida RC-4(pSI30) or its parent strain were least preferred, but microflagellates did not exhibit consistent preferences. Fluorescent antibodies were prepared against isolates to permit detection of target cells in natural communities. In three different microcosm experiments the cell densities of introduced isolates declined over a period of days. In one experiment, 3-CB additions (100 mg/L) led to increases of P. alcaligenes C-0 and P. acidovorans M3GY cell densities within 1 day, although P. putida RC-4(pSI30) took 4 days. In a second experiment, the persistence of P. putida RC-4(pSI30) and its parent strain P. putida RC-4 were compared and rates of initial population decline were not statistically different. 3-CB addition stimulated the growth of other organisms while densities of the P. putida strains further declined. In a third experiment exposure to 100 mg 3-CB/L slowed the rate of decline of P. acidovorans M3GY densities compared to a 10 mg/L concentration or unamended control. Competition with the native flora was a significant factor affecting the persistence of introduced 3-CB degraders.

The genetic regulation of liver microsomal CYP2E1 activity among strains of the viviparous fish Poeciliopsis.

CYP2E1 expression was examined within, among, and in F(1) and backcross progeny of strains (P. monacha S68-5; P. viriosa M65-23) of the viviparous fish Poeciliopsis. CYP 2E1 activity varied dramatically in P. monacha, and P. viriosa (3.9+/-0.8 and 9.6+/-1.3 microg/min/mg) as well as the temperature which gave maximal activity (T(0)=25 degrees C and 31 degrees C). F(1) individuals from a crosses between P. monacha and P. viriosa, produced progeny whose CYP2E1 activity segregated into three different groups: (1) phenotypically the same as P. viriosa; (2) intermediate between the two parental strains; and (3) phenotypically the same as P. monacha. When a male P. monacha was crossed with a female P. viriosa 25% of the offspring had an intermediate phenotype and 65% the maternal P. viriosa phenotype. From the same cross, 85% of the females progeny had the maternal phenotype, while 80% of male progeny had the intermediate and paternal phenotype, suggesting an effect of the maternal genome on the F(1) phenotype. Among F(1) fish the T(0) was evenly distributed between parental values. In the backcross of a F(1) female to a male P. viriosa, CZX-6-hydroxylase activity segregated into the same three phenotypes with 60% of the progeny expressing the P. monacha phenotype. From the same cross, 70% of females and 40% of males expressed the P. monacha phenotype. The T(0) in the backcross were evenly distributed between the two parental values and the sex ratio among progeny was different than expected.

Improved analysis of dissolved carbohydrates in stream water with HPLC-PAD.

This paper describes improvements made to the determination of dissolved carbohydrates in stream water with high-performance liquid chromatography and pulsed amperometric detection (HPLC-PAD). We eliminated interference from dissolved oxygen, separated xylose and mannose along with other molecules, reduced the chromatographic peak shift associated with carbonate accumulation in the column to less than 1% for all samples, and achieved an 8% increase in recovery of hydrolyzed carbohydrates by replacing ion-exchange desalting cartridges with evaporative removal of HCl under N2. These modifications lowered detection limits to less than or equal to 0.4 nM for 10 different monosaccharides and enhanced our ability to follow the dynamics of these molecules in stream ecosystems. In the determination of dissolved free monosaccharides, average relative precision was 1.3% and recovery ranged from 92 to 109%. For dissolved total saccharides, average relative precision was 3.3%. Concentrations were stable when filtered (0.2 microm) stream water was stored for 1 day at 22 degrees C, 1 week at 4 degrees C, or 1.5 months at -20 degrees C.

Killifish metallothionein messenger RNA expression following temperature perturbation and cadmium exposure.

Metallothionein (MT), a cysteine-rich metal binding protein, is considered to play an essential role in the regulation of intracellular metals. Induction of MT in mammalian and nonmammalian tissues following heavy metal exposure may serve as a defense mechanism and a biomarker of environmental exposure to chemical stressors such as toxic metals. In this study, MT messenger RNA (mRNA) expression was characterized in male and female nonspawning and spawning killifish (Fundulus heteroclitus) following an 8-day exposure to specific sublethal stressors, which included temperature perturbation (26 degrees C or 10 degrees C) and/or 6 ppb of waterborne cadmium chloride (CdCl2). Hepatic, gill, and intestinal MT mRNA, expressed as copy number per microgram of total RNA, was assessed by reverse transcriptase-polymerase chain reaction and electrochemiluminescence using winter flounder (Pleuronectes americanus) MT complementary DNA primers. Liver, gill, and intestine MT mRNA expression was significantly (P < 0.05) increased in nonspawning killifish exposed to 26 degrees C compared with those exposed to 19 degrees C (control). In addition, a significant (P < 0.05) increase in gill MT mRNA induction was observed in nonspawning killifish exposed to 6 ppb of waterborne CdCl2 compared with controls. The results of this study demonstrate significant MT mRNA induction in nonspawning killifish following short-term exposure to physiological and chemical stressors. Thus, further research may be necessary before the use of killifish MT mRNA induction as a biomarker of environmental chemical stress exposure alone.

Multiple pathways of prostate carcinogenesis analyzed by using cultured cells isolated from rats treated with N-methyl-N-nitrosourea and testosterone.

Treatment of rats with N-methyl-N-nitrosourea (MNU) and testosterone results in a high incidence of metastasizing dorsolateral prostate tumors. In previous studies, a high frequency (> or = 70%) of a G35 --> A transition mutation at the second position of codon 12 of the Ki-ras oncogene was found in these tumors. This was confirmed in the study reported here, and the frequency of this mutation appeared similar in tumors induced in four different rat strains, regardless of differences in sensitivity among these strains to the induction of prostate cancers by MNU and testosterone: Wistar Furth (62% incidence of grossly visible prostate tumors) > Lobund Wistar (55%) > Fisher 344 (40%) > Copenhagen (37%). A method was developed to isolate and separately culture epithelial and stromal cells from these rat prostate carcinomas. Of 20 primary cell cultures established from histologically confirmed rat prostate carcinomas, 19 (95%) displayed one or more of the following characteristics: the Ki-ras mutation (17 of 20; 85%), anchorage-independent growth in soft agar at early passage (12 of 20; 60%), or tumorigenicity at later passage (eight of eight; 100%). One epithelial cell culture and all five stromal cell cultures established from prostate tumors had none of these characteristics. Epithelial cultures that had the Ki-ras mutation and grew in soft agar constitute the predominant genotype/phenotype (55%), cultures with the mutation that did not grow in soft agar were less frequent (30%), 10% of the cultures had neither characteristic, and only one grew in soft agar but did not have the mutation. These findings suggest that there are at least two and perhaps more different molecular pathways of prostate carcinogenesis in rats treated with MNU plus testosterone. Furthermore, these data suggest that these pathways and the mechanisms determining strain differences in sensitivity to prostate cancer induction are unrelated.

Determination and application of desirable analytical performance goals: the ISO/TC 212 approach.

Desirable analytical performance goals are needed to place clinical laboratories' quality management plan on a rational basis. While the most important basis of desirable analytical performance goals is medical needs, a well-established quality management plan must take into account economic and regulatory needs. There are many approaches that have been proposed for determining desirable analytical performance goals. The ISO/TC 212 approach for rationalizing the various approaches for determining desirable analytical performance goals, which is based on the ability to meet medical needs, is presented.

Insect repellents. What really works?

DEET remains one of the most effective repellents against a wide variety of insects. Although adverse reactions have been reported in the medical literature and magnified in the press, the compound is remarkably safe and has been used by hundreds of millions of people over the past 40 years. Permethrin is a better deterrent of ticks and, like DEET, is remarkably safe. Concomitant use of these two agents provides superior protection. Citronella and a bath oil, Avon Skin-So-Soft, also provide limited protection against some types of flying insects. The promise of new agents or protective strategies is on the horizon. Recently it was shown that retroviral vectors could be used to integrate and express foreign genes in the malaria mosquito, Anopheles gambiae. Conceivably, a genetically engineered mosquito that is resistant to malaria and other transmissible diseases may one day be developed, obviating some of the need for repellents. Almost certainly, future research will yield additional agents to further protect against mosquitoes.

Nausea and vomiting after gynaecological surgery: a meta-analysis of factors affecting their incidence.

A total of 1442 patients who had major gynaecological surgery were recruited into three multicentre studies using a standard general anaesthetic technique in order to assess the efficacy of various doses of orally administered ondansetron in the prevention of postoperative nausea and vomiting. A total of 1257 patients were included in this analysis: 420 received oral formulations of placebo and 212, 296 and 329 received ondansetron 1, 8 and 16 mg, respectively. The following factors were measured in these studies and were considered to have a possible influence on the proportion of patients experiencing postoperative nausea and vomiting: age of patient; volatile anaesthetic; intraoperative dose of fentanyl; postoperative dose of morphine; country; anaesthesia duration; neuromuscular blocker; neuromuscular block antagonist; premedicant; recovery time; type of surgery; antiemetic treatment; body weight. Using a process of elimination based on logistic regression techniques, the factors found to be the most important influences on the frequencies of nausea and vomiting were antiemetic treatment, type of surgery, neuromuscular blocker, country, volatile anaesthetic and age. A statistically significant interaction between type of surgery and age was observed. Adjusted probabilities of nausea and vomiting were obtained from the model, including all the above factors, together with the type of surgery by age interaction. Ondansetron 8 mg showed the smallest adjusted probability of nausea (0.54) and vomiting (0.34) and placebo the greatest (nausea 0.75, vomiting 0.61). A similar pattern of adjusted probabilities over neuromuscular blocking agents was seen for nausea and vomiting, with the greatest occurring in patients receiving pancuronium (nausea 0.74, vomiting 0.57) and the least in patients receiving alcuronium (nausea 0.59, vomiting 0.38).(ABSTRACT TRUNCATED AT 250 WORDS)

Evaluation and simplification of the assimilable organic carbon nutrient bioassay for bacterial growth in drinking water.

A modified assimilable organic carbon (AOC) bioassay is proposed. We evaluated all aspects of the AOC bioassay technique, including inoculum, incubation water, bioassay vessel, and enumeration technique. Other concerns included eliminating the need to prepare organic carbon-free glassware and minimizing the risks of bacterial and organic carbon contamination. Borosilicate vials (40 ml) with Teflon-lined silicone septa are acceptable incubation vessels. Precleaned vials are commercially available, and the inoculum can be injected directly through the septa. Both bioassay organisms, Pseudomonas fluorescens P-17 and Spirillum sp. strain NOX, are available from the American Type Culture Collection and grow well on R2A agar, making this a convenient plating medium. Turbid raw waters need to be filtered prior to an AOC analysis. Glass fiber filters used with either a peristaltic pump or a syringe-type filter holder are recommended for this purpose. A sampling design that emphasizes replication of the highest experimental level, individual batch cultures, is the most efficacious way to reduce the total variance associated with the AOC bioassay. Quality control for the AOC bioassay includes an AOC blank and checks for organic carbon limitation and inhibition of the bioassay organisms.

Development of a rapid assimilable organic carbon method for water.

A rapid method for measurement of assimilable organic carbon (AOC) is proposed. The time needed to perform the assay is reduced by increasing the incubation temperature and increasing the inoculum density. The ATP luciferin-luciferase method quickly enumerates the test organisms without the need for plate count media or dilution bottles. There was no significant difference between AOC values determined with strain P17 for the ATP and plate count procedures. For strain NOX, the plate count procedure underestimated bacterial levels in some samples. Comparison of AOC values obtained by the Belleville laboratory (by the ATP technique) and the Stroud Water Research Center (by plate counts) showed that values were significantly correlated and not significantly different. The study concludes that the rapid AOC method can quickly determine the bacterial growth potential of water within 2 to 4 days.

The effect of oral ondansetron in the prevention of postoperative nausea and vomiting after major gynaecological surgery performed under general anaesthesia.

The efficacy and safety of ondansetron in preventing postoperative nausea and vomiting following major gynaecological surgery was evaluated in this multicentre, double-blind study. A total of 243 patients were randomised to receive three doses of oral ondansetron 8 mg or matching placebo at 8-hourly intervals, with the first dose being given an hour before surgery. A standard general anaesthetic technique was employed throughout. Nausea, vomiting and safety assessments were performed continuously during the 24 h postrecovery period. Of the 237 patients evaluated for efficacy, significantly fewer ondansetron 8 mg treated patients (65/117; 56%) experienced postoperative nausea and/or vomiting compared with placebo-treated patients (94/120; 78%) during the study period (p < 0.001). In addition, ondansetron 8 mg reduced the severity of nausea (p < 0.001) and the total number of vomiting episodes experienced (p < 0.001). Overall, ondansetron 8 mg was well tolerated and effective in preventing postoperative nausea and vomiting in this surgical setting.

The clinical development of ondansetron for use in the prevention and treatment of postoperative nausea and vomiting.

An international clinical trial programme has been established to assess the efficacy and safety of ondansetron in the prevention and treatment of postoperative nausea and vomiting. The programme included nine pilot studies and six key placebo-controlled studies. These studies have evaluated both oral and intravenous formulations of ondansetron in the prevention of postoperative nausea and vomiting, and intravenously administered ondansetron in the treatment of established symptoms. Most patients included in the trials were adult women, less than 50 years of age, receiving anaesthesia for gynaecological surgery. The primary efficacy analysis for emesis was based on the assessment of complete response (i.e. absence of emetic episodes or nausea in the first 24 h postoperatively). These trials clearly demonstrated the anti-emetic efficacy of ondansetron in the prevention and treatment of postoperative nausea and vomiting.

Assessment of [h]thymidine incorporation into DNA as a method to determine bacterial productivity in stream bed sediments.

We performed several checks on the underlying assumptions and procedures of the thymidine technique applied to stream bed sediments. Bacterial production rates were not altered when sediments were mixed to form a slurry. Incubation temperature did affect production rates. Controls fixed and washed with formaldehyde had lower backgrounds than trichloroacetic acid controls. DNA extraction by base hydrolysis was incomplete and variable at 25 degrees C, but hydrolysis at 120 degrees C extracted 100% of the DNA, of which 84% was recovered upon precipitation. Production rates increased as thymidine concentrations were increased over 3 orders of magnitude (30 nM to 53 muM thymidine). However, over narrower concentration ranges, thymidine incorporation into DNA was independent of thymidine concentration. Elevated exogenous thymidine concentrations did not eliminate de novo synthesis. Transport of thymidine into bacterial cells occurred at least 5 to 20 times faster than incorporation of label into DNA. We found good agreement between production rates of bacterial cultures based upon increases in cell numbers and estimates based upon thymidine incorporation and amount of DNA per cell. Those comparisons emphasized the importance of isotopic dilution measurements and validated the use of the reciprocal plot technique for estimating isotopic dilution. Nevertheless, the thymidine technique cannot be considered a routine assay and the inability to measure the cellular DNA content in benthic communities restricts the accuracy of the method in those habitats.