Temperature-dependent physiological and biochemical responses of the marine medaka Oryzias melastigma with consideration of both low and high thermal extremes.

This study aimed to investigate temperature effect on physiological and biochemical responses of the marine medaka Oryzias melastigma larvae. The fish were subjected to a stepwise temperature change at a rate of 1 °C/h increasing or decreasing from 25 °C (the control) to six target temperatures (12, 13, 15, 20, 28 and 32 °C) respectively, followed by a 7-day thermal acclimation at each target temperature. The fish were fed ad libitum during the experiment. The results showed that cumulative mortalities were significantly increased at low temperatures (12 and 13 °C) and at the highest temperature (32 °C). For the survivors, their growth profile closely followed the left-skewed 'thermal performance curve'. Routine oxygen consumption rates of fish larvae were significantly elevated at 32 °C but suppressed at 13 and 15 °C (due to a high mortality, larvae from 12 °C were not examined). Levels of heat shock proteins and activities of malate dehydrogenase and lactate dehydrogenase were also measured in fish larvae exposed at 15, 25 and 32 °C. The activities of both enzymes were significantly increased at both 15 and 32 °C, where the fish larvae probably suffered from thermal discomfort and increased anaerobic components so as to compensate the mismatch of energy demand and supply at these thermal extremes. Coincidently, heat shock proteins were also up-regulated at both 15 and 32 °C, enabling cellular protection. Moreover, the critical thermal maxima and minima of fish larvae increased significantly with increasing acclimation temperature, implying that the fish could develop some degrees of thermal tolerance through temperature acclimation.

Construction by gene targeting in human cells of a "conditional' CDC2 mutant that rereplicates its DNA.

We describe a novel gene targeting strategy for the genetic analysis of essential genes in mammalian cells and its use to study the role of the cell cycle control gene CDC2 in human cells. A cell line (HT2-19) was generated in which endogenous CDC2 gene expression and cell viability depend on the presence of an inducer in the growth medium. In the absence of inducer, HT2-19 cells undergo extensive DNA rereplication and apoptosis. Rereplication is indicative of a role for human CDC2 in a control mechanism, previously undetected in mammalian cells, that prevents premature entry into S-phase.

Extreme cold or warm events can potentially exacerbate chemical toxicity to the marine medaka fish Oryzias melastigma.

Marine ecosystems are currently subjected to dual stresses of chemical pollution and climate change. Through a series of laboratory experiments, this study investigated the impact of exposure to chemical contaminant such as DDT or copper (Cu), in combination with cold or warm temperature extremes on the marine medaka fish Oryzias melastigma. The results showed that extreme seawater temperatures (i.e., 15 and 32 °C in sub-tropical Hong Kong) exacerbated adverse chemical impacts on the growth performance of O. melastigma, in particular at the high thermal extreme. This was likely associated with an interruption of oxygen consumption and aerobic scope. Most importantly, the results of acclimation experiments, as reflected by thermal tolerance polygons, showed that chemical exposure substantially narrowed the thermal tolerance of the medaka, making them more vulnerable to temperature changes and extreme thermal events. Under dual stresses of thermal extremes and chemical exposure, the medaka switched their metabolic pathway to anaerobic respiration that might deplete their energy reserve for chemical detoxification. Although stress proteins such as heat shock proteins (HSP90) were up-regulated for cellular protection in the fish, such a defensive mechanism was repressed with intensifying dual stresses at high temperature and high chemical concentration. Bioconcentration of DDT or Cu generally increased with increasing temperature and its exposure concentration. Overall, these complex chemical-temperature interactions concomitantly exerted a concerted adverse impact to O. melastigma. The temperature-dependent toxicity of DDT or Cu shown in this study clearly demonstrated the potential challenge brought by the risk of chemical pollution under the impact of global climate change.

Inhibition of Ced-3/ICE-related proteases does not prevent cell death induced by oncogenes, DNA damage, or the Bcl-2 homologue Bak.

There is increasing evidence for a central role in mammalian apoptosis of the interleukin-1 beta-converting enzyme (ICE) family of cysteine proteases, homologues of the product of the nematode "death" gene, ced-3. Ced-3 is thought to act as an executor rather than a regulator of programmed cell death in the nematode. However, it is not known whether mammalian ICE-related proteases (IRPs) are involved in the execution or the regulation of mammalian apoptosis. Moreover, an absolute requirement for one or more IRPs for mammalian apoptosis has yet to be established. We have used two cell-permeable inhibitors of IRPs, Z-Val-Ala-Asp.fluoromethylketone (ZVAD.fmk) and t-butoxy carbonyl-Asp.fluoromethylketone (BD.fmk), to demonstrate a critical role for IRPs in mammalian apoptosis induced by several disparate mechanisms (deregulated oncogene expression, ectopic expression of the Bcl-2 relative Bak, and DNA damage-induced cell death). In all instances, ZVAD.fmk and BD.fmk treatment inhibits characteristic biochemical and morphological events associated with apoptosis, including cleavage of nuclear lamins and poly-(ADP-ribose) polymerase, chromatin condensation and nucleosome laddering, and external display of phosphatidylserine. However, neither ZVAD.fmk nor BD.fmk inhibits the onset of apoptosis, as characterized by the onset of surface blebbing; rather, both act to delay completion of the program once initiated. In complete contrast, IGF-I and Bcl-2 delay the onset of apoptosis but have no effect on the kinetics of the program once initiated. Our data indicate that IRPs constitute part of the execution machinery of mammalian apoptosis induced by deregulated oncogenes, DNA damage, or Bak but that they act after the point at which cells become committed to apoptosis or can be rescued by survival factors. Moreover, all such blocked cells have lost proliferative potential and all eventually die by a process involving cytoplasmic blebbing.

Thermal extremes can intensify chemical toxicity to freshwater organisms and hence exacerbate their impact to the biological community.

Temperature in freshwater ecosystems fluctuates daily, seasonally and yearly. Climate change further induces temperature variations. In this study, we hypothesise that water temperatures, in particular thermal extremes, can significantly influence chemical toxicity to ectothermic organisms. Although temperature-dependent chemical toxicity (TDCT) is a classic research area in ecotoxicology, a unified model for predicting TDCT for freshwater species is yet to be developed. This study aimed to address this challenging issue through a meta-analysis by comparing acute toxicity endpoints (i.e. median lethal or effective concentration data; LC50 or EC50) of 13 chemicals for various freshwater species generated from different temperatures. Our results suggest that in most cases, freshwater species exhibit the highest tolerance towards chemicals at their physical optimal temperature (Topt), and chemical toxicity exacerbates when temperature is higher or lower than Topt (i.e. inverted V-shaped model between temperature and LC50 or EC50). Such observations are further supported by temperature-dependent hazardous concentration 10% (HC10) values derived from species sensitivity distributions constructed using toxicity data generated at different temperatures. A unified mathematical model was also developed to describe the inverted V-shape relationship between temperature and HC10 derivations. Overall, considering the natural variations of freshwater temperatures, the inverted V-shaped TDCT model can be readily applied to derive water quality guidelines and assess ecological risks of chemical contaminants.

Deriving site-specific sediment quality guidelines for Hong Kong marine environments using field-based species sensitivity distributions.

Field data of benthic communities and contaminant loadings in marine sediments measured in parallel can be used to derive sediment quality guidelines (SQGs) using a field-based species sensitivity distribution (f-SSD) approach. Recently, SQGs have been successfully derived from f-SSDs for the Norwegian continental shelf with an extensive survey (>1 million km(2)) and a large data set (1,902 sampling stations with 1,944 species). The present study examined the practicality of this approach in deriving SQGs for a much smaller geographical area, namely, the marine environment of Hong Kong (sea area: 1,651 km(2)), making use of databases of the government of Hong Kong special administrative region. As the construction of f-SSDs requires the use of a collection of responses from individual species to a chemical gradient in sediment, data screening criteria on the minimum abundance of the species were evaluated and optimized to ensure sufficient statistical power for estimating these responses. Sediment quality guidelines were derived for nine trace metals, total polycyclic aromatic hydrocarbons, and total polychlorinated biphenyls and compared with current SQGs in developed countries. The community-adjusted hazardous concentrations of 5% and 10% of the f-SSDs were adopted to represent the threshold effects level (TEL) and predicted effects level (PEL), respectively. The TELs derived from this f-SSD approach compares favorably with current SQGs, while the derived PELs were generally lower than the current SQGs, indicating that they are more protective. The f-SSDs can be directly utilized for probabilistic risk assessment, while the field-based SQGs can be used as site-specific guidelines or integrated into current SQGs. Our results suggest that the f-SSD approach can also be applicable to small areas such as Hong Kong.

Synergistic toxic effects of zinc pyrithione and copper to three marine species: Implications on setting appropriate water quality criteria.

Zinc pyrithione (ZnPT) is widely applied in conjunction with copper (Cu) in antifouling paints as a substitute for tributyltin. The combined effects of ZnPT and Cu on marine organisms, however, have not been fully investigated. This study examined the toxicities of ZnPT alone and in combination with Cu to the diatom Thalassiosira pseudonana, polychaete larvae Hydroides elegans and amphipod Elasmopus rapax. Importantly, ZnPT and Cu resulted in a strong synergistic effect with isobologram interaction parameter lambda>1 for all test species. The combined toxicity of ZnPT and Cu was successfully modelled using the non-parametric response surface and its contour. Such synergistic effects may be partly due to the formation of copper pyrithione. It is, therefore, inadequate to assess the ecological risk of ZnPT to marine organisms solely based on the toxicity data generated from the biocide alone. To better protect precious marine resources, it is advocated to develop appropriate water quality criteria for ZnPT with the consideration of its compelling synergistic effects with Cu at environmentally realistic concentrations.

Cytochemistry and ultrastructural morphometry of cultured HL60 myeloid leukemia cells.

Cultured human myeloid leukemia (HL60) cells were characterized using ultrastructural cytochemical methods and differences identified when cells were compared for low (17 to 47), middle (69 to 100), and high (214 to 244) passages or to normal promyelocytes aspirated from bone marrow. Endoplasmic reticulum and transition structures (pre-Golgi compartment) of HL60 cells stained positively for peroxidase using diaminobenzidine but stained sparsely for reducing groups with osmium-zinc iodide. Staining of Golgi elements was relatively indistinct with diaminobenzidine and strong with osmium-zinc iodide, in comparison to freshly harvested promyelocytes which have intense diaminobenzidine and osmium-zinc iodide staining of the pre-Golgi and Golgi compartments. Cytoplasmic polyribosomes were more numerous in middle and high passage cells, whereas dilatation of endoplasmic reticulum was less prominent in these cells. The mean granule size was significantly increased in low passage cells, and staining of peroxidase was more prominent by light and electron microscopy when compared to high passage cells. Cytoplasmic granules demonstrated strong complex carbohydrate staining, indicating a lack of granule maturation in HL60 cells. Terminally differentiated myeloid cells were more frequent in low passage samples, and some neutrophil granule maturation appeared to occur within these cells, whereas all eosinophil granules consistently remained immature with intense complex carbohydrate staining and lack of crystalloid formation. These studies demonstrate significant differences between HL60 cells and normal promyelocytes, and also passage-dependent maturational differences in HL60 cells. These differences should be considered in evaluating parameters of cell growth and maturation and in the biochemical and enzymatic characterization of these cells.

Heterogeneity of peroxidase-positive granules in normal human an Chédiak-Higashi neutrophils.

Studies have demonstrated significant heterogeneity in neutrophil granule morphology and physical density. This study evaluated the heterogeneity morphometrically, morphologically, cytochemically, and biochemically. Intact human peripheral blood neutrophils collected from normal volunteers and a patient with Chédiak-Higashi syndrome (CHS) and isolated normal neutrophil granules were processed for ultrastructural morphology and peroxidase staining. Intact cells, nuclei, and granule profiles were analyzed by computer-assisted planimetry. Peroxidase-positive granules (PPG) represented about 40% of normal neutrophil granules and covered the entire spectrum of granule size. PPG in the least-dense fractions of isolated granules were significantly smaller than in higher-density fractions. PPG in low- and intermediate-density fractions differed from high-density fraction by moderate to strong vicinal glycol staining with Thiéry's periodate-thiocarbohydrazide-silver proteinate method. Differing ratios of % beta-glucuronidase/% myeloperoxidase (MPO) across granule fractions indicated PPG heterogeneity. Morphometric analysis of neutrophils treated with 1 microM calcium ionophore A23187 did not show significant differences in PPG size or number. Biochemically analyzed MPO in these cells was preserved, although the number of peroxidase-negative granules (PNG) and levels of vitamin B12-binding protein were markedly decreased. In CHS, about 20% of granules were PPG. Analysis of CHS neutrophils revealed the persistence of microgranules similar to normals. PNG number and volume fractions of PPG and TG were not different from normals. Complex heterogeneity of normal PPG was quantitated using morphometry and appeared preserved in ionophore-treated cells but was uniquely modified in CHS.

Ultrastructural silver enhancement of Prussian blue-reactive iron in hematopoietic and intestinal cells.

Prussian blue has been widely used to localize iron in a variety of tissues at the light and electron microscopic level. In the present study, thin sections of human marrow and blood cells and rat duodenal cells were exposed to silver proteinate (SP) after staining en bloc with acid ferrocyanide (AF), with and without prior iron saturation using iron nitrilotriacetate (FeNTA). Silver deposition was observed over Prussian blue-reactive sites and significantly enhanced sites of minimal AF and FeNTA-AF staining. AF-SP stain deposits were present in the cytoplasmic matrix, granules, and occasionally on the surfaces of macrophages, monocytes, and erythroblasts. FeNTA-AF-SP stained additional cytoplasmic and surface sites in erythroblasts and stained neutrophil granules intensely. Duodenal epithelium from iron-loaded rats demonstrated strong AF-SP staining of ferric iron in microvilli, apical cytoplasmic matrix, and lateral membranes. Similar preparations from iron-replete rats stained sparsely; however, intense AF-SP staining was observed after iron saturation with FeNTA. SP similarly enhanced luminal ferrous iron deposits stained with acid ferricyanide in rats given intraluminal ferrous iron. AF-SP stain deposits were removed by exposure of thin sections to NH4OH, KCN, or HNO3 but were not affected by prior exposure to HIO4 or NaBH4, consistent with a silver cyanide or complex stain precipitate rather than reduced silver or silver ferriferrocyanide. SP enhancement of Prussian blue allows identification of reactive sites not readily visualized with AF or FeNTA-AF alone, and offers the potential for differentiating AF staining from other deposits or organelles of comparable density.

Geographic variation in exercise testing by family physicians.

BACKGROUND: The purposes of this study were to determine the extent to which exercise stress testing is performed by family physicians; whether rural physicians are more likely to utilize exercise stress testing than their urban counterparts; and what factors influence their decisions. METHODS: A random sample of 211 practicing members of the Nebraska Academy of Family Physicians was surveyed. Responses were received from 163 (77%). To ensure independence, if two or more subjects were members of the same group practice, one was randomly assigned to the study, for a total of 125 respondents available for analysis. Questionnaire items included performance of exercise stress tests, population base, and distance to the nearest specialist who performed the test. Respondents were classified as urban, rural, or frontier, based on population per square mile in their county. RESULTS: Seventy-three of the 125 respondents (58%) reported that they perform exercise stress testing. Physicians in rural or frontier counties were twice as likely to perform the test as urban physicians (P < .001). Similar results were found for distance to the closest specialist who performs exercise stress tests (P < .001) and reported population base (P < .01). Of those performing the procedure, 42 (58%) indicated they had learned it during residency, whereas 15 (21%) were self-taught or had learned from a colleague. CONCLUSIONS: Family physicians in rural Nebraska are significantly more likely to perform exercise stress testing than those in urban areas and much more likely to do stress testing than previous national studies indicate. National guidelines should acknowledge the need for family physicians to perform exercise tests and promote training in this procedure.

Improving smoking cessation counseling by family practice residents.

BACKGROUND: While programs to train residents in smoking cessation counseling skills have been devised, few have assessed trainee behavioral changes in practice settings where residents were blind to the evaluation of their behavior. This study assessed the effectiveness of a training program in smoking cessation counseling and chart-prompting system in increasing the frequency and quality of counseling by residents at three clinic sites. METHODS: Twenty-eight residents participated in a training program that included epidemiology, discussion of attitudes, counseling techniques, videotaped examples, and small group role play. The chart-prompting system was implemented at two clinics 1 month after training. Patient exit interviews, during which information on resident counseling on smoking cessation was obtained, were conducted before training, after training, at 3-month follow-up, and at 6-month follow-up. Questionnaires assessing knowledge, attitudes, and self-perceived counseling behaviors were completed by residents at pretraining, posttraining, and 6-month follow-up periods. RESULTS: Interviews with 517 smokers were analyzed. Results showed an increase in counseling at 3-month follow-up but a regression toward baseline at 6 months. Counseling improved at clinics where chart prompting was initiated. The number of counseling behaviors decreased when the number of patients seen increased. Whether a patient received counseling was positively associated with prior contact with the physician. There was no correlation between resident self-perception and patient report. CONCLUSIONS: A training program in smoking cessation counseling and a chart-prompting system did not result in a lasting change in resident behavior. System factors may play an important role in long-term behavior change.

An ounce of prevention? Evaluation of the 'Put Prevention into Practice' program.

BACKGROUND: The "Put Prevention into Practice" (PPIP) program was designed to enhance the capacity of health care providers to deliver clinical preventive services. This study was designed to evaluate the program's effectiveness when applied to family physicians in private practice settings. METHODS: Eight Midwestern practices that had purchased PPIP kits were identified and agreed to participate in the study. A comparative case study approach encompassing a variety of data collection techniques was used. These techniques included participant observation of clinic operations and patient encounters, semistructured and key informant interviews with physicians and staff members, chart reviews, and structured postpatient encounter and office environment checklists. Content analysis of the qualitative data and construction of the individual cases were done by consensus of the research team. RESULTS: PPIP materials are not being used, even by the clinics that ordered them. Physicians already providing quality preventive services prefer their existing materials to those in the PPIP kit. Sites that are underutilizing preventive services are unable or unwilling to independently implement the PPIP program. CONCLUSIONS: Development of technical support may facilitate implementation of PPIP materials into those practices most deficient in providing preventive services. Given the diversity of practice environments it is unlikely that a "one size fits all" approach will ever be able to address the needs of all providers.

Deriving field-based sediment quality guidelines from the relationship between species density and contaminant level using a novel nonparametric empirical Bayesian approach.

This paper describes a novel statistical approach to derive ecologically relevant sediment quality guidelines (SQGs) from field data using a nonparametric empirical Bayesian method (NEBM). We made use of the Norwegian Oil Industrial Association database and extracted concurrently obtained data on species density and contaminant levels in sediment samples collected between 1996 and 2001. In brief, effect concentrations (ECs) of each installation (i.e., oil platform) at a given reduction in species density were firstly derived by fitting a logistic-type regression function to the relationship between the species density and the corresponding concentration of a chemical of concern. The estimated ECs were further improved by the NEBM which incorporated information from other installations. The distribution of these improved ECs from all installations was determined nonparametrically by the kernel method, and then used to determine the hazardous concentration (HC) which can be directly linked to the species loss (or the species being protected) in the sediment. This method also enables an accurate estimation of the lower confidence limit of the HC, even when the number of observations was small. To illustrate the effectiveness of this novel technique, barium, cadmium, chromium, copper, mercury, lead, tetrahydrocannabinol, and zinc were chosen as example contaminants. This novel approach can generate ecologically sound SQGs for environmental risk assessment and cost-effectiveness analysis in sediment remediation or mud disposal projects, since sediment quality is closely linked to species density.

Acute and chronic toxicities of zinc pyrithione alone and in combination with copper to the marine copepod Tigriopus japonicus.

Zinc pyrithione (ZnPT) is a widely used booster biocide in combination with copper (Cu) in antifouling paints as a substitute for tributyltin. The co-occurrence of ZnPT and Cu in coastal marine environments is therefore very common, and may pose a higher risk to marine organisms if they can result in synergistic toxicity. This study comprehensively investigated the combined toxicity of ZnPT and Cu, on the marine copepod Tigriopus japonicus, for the first time, based on both 96-h acute toxicity tests using adult copepods and chronic full-life cycle tests (21 d) using nauplii <24-h old. As ZnPT has been reported to be easily trans-chelated to copper pyrithione (CuPT) in the presence of Cu, the acute toxicities of CuPT alone and in combination with Cu on adult copepods were also assessed. Our results showed that ZnPT and Cu exhibited a strong synergistic toxic effect on the copepod in both acute and chronic tests. During the acute test, the mortalities of adult copepods increased dramatically even with an addition of Cu at concentrations as low as 1-2 µg/L compared with those exposed to ZnPT alone. Severe chronic toxicities were further observed in the copepods exposed to ZnPT-Cu mixtures, including a significant increase of naupliar mortality, postponing of development from naupliar to copepodid and from copepodid to adult stage, and a significant decrease of intrinsic population growth when compared with those of copepods exposed to ZnPT or Cu alone. Such synergistic effects might be partly attributable to the formation of CuPT by the trans-chelation of ZnPT and Cu, because CuPT was found to be more toxic than ZnPT based on the acute toxicity results. Mixtures of CuPT and Cu also led to synergistic toxic effects to the copepod, in particular at high Cu concentrations. A novel non-parametric response surface model was applied and it proved to be a powerful method for analysing and predicting the acute binary mixture toxicities of the booster biocides (i.e., ZnPT and CuPT) and Cu on the copepod. To better protect precious marine resources, it is necessary to revise and tighten existing water quality criteria for biocides, such as ZnPT and CuPT, to account for their synergistic effects with Cu at environmentally realistic levels.

The difference between temperate and tropical saltwater species' acute sensitivity to chemicals is relatively small.

Due to a lack of saltwater toxicity data in tropical regions, toxicity data generated from temperate or cold water species endemic to North America and Europe are often adopted to derive water quality guidelines (WQG) for protecting tropical saltwater species. If chemical toxicity to most saltwater organisms increases with water temperature, the use of temperate species data and associated WQG may result in under-protection to tropical species. Given the differences in species composition and environmental attributes between tropical and temperate saltwater ecosystems, there are conceivable uncertainties in such 'temperate-to-tropic' extrapolations. This study aims to compare temperate and tropical saltwater species' acute sensitivity to 11 chemicals through a comprehensive meta-analysis, by comparing species sensitivity distributions (SSDs) between the two groups. A 10 percentile hazardous concentration (HC10) is derived from each SSD, and then a temperate-to-tropic HC10 ratio is computed for each chemical. Our results demonstrate that temperate and tropical saltwater species display significantly different sensitivity towards all test chemicals except cadmium, although such differences are small with the HC10 ratios ranging from 0.094 (un-ionised ammonia) to 2.190 (pentachlorophenol) only. Temperate species are more sensitive to un-ionised ammonia, chromium, lead, nickel and tributyltin, whereas tropical species are more sensitive to copper, mercury, zinc, phenol and pentachlorophenol. Through comparison of a limited number of taxon-specific SSDs, we observe that there is a general decline in chemical sensitivity from algae to crustaceans, molluscs and then fishes. Following a statistical analysis of the results, we recommend an extrapolation factor of two for deriving tropical WQG from temperate information.

Acute and chronic toxicities of Irgarol alone and in combination with copper to the marine copepod Tigriopus japonicus.

Irgarol 1051 has been widely used as a booster biocide in combination with copper (Cu) in antifouling paints. The combined toxicity of Irgarol with Cu on marine organisms, however, has not been fully investigated. This study investigated the acute and chronic toxicities of binary mixtures of Irgarol and CuSO(4) to the marine copepod Tigriopus japonicus. The acute combined toxicity of Irgarol and Cu was simple additive as revealed by two response surface models and their contours. However, based on chronic full life-cycle tests, when Irgarol was combined with Cu at an environmentally realistic concentration (10 µg L(-1)), a slightly synergistic effect was observed at a high Irgarol concentration (940 µg L(-1)), as shown by a significant increase in larval mortality. As Cu contamination is widespread in coastal environments, our results entail the importance of considering the combined toxic effect of the booster biocide and Cu for setting ecologically realistic water quality criteria.