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.

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.

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.

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.

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.

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.

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.

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.

Use of field data to support European Water Framework Directive quality standards for dissolved metals.

Quality standards (QS) for dissolved metals in freshwaters have been proposed underthe European Water Framework Directive (WFD) and are based mainly upon laboratory ecotoxicity data. Uncertainties remain about laboratory-to-field extrapolation to establish QS that are neither over- nor underprotective. Freshwater benthic macroinvertebrates are a group of organisms of known sensitivity to heavy metals. We analyzed a dataset from England and Wales of dissolved metal concentrations (cadmium, chromium, copper, iron, nickel, lead, and zinc) and associated benthic invertebrate community metrics, using piecewise regression, quantile regression, and information on metal concentrations consistent with good quality status. Analysis of these field data suggests that dissolved metal QS proposed under the WFD are similar to metal concentrations in rivers associated with unimpaired benthic invertebrate assemblages in England and Wales. The only exceptions to this are QS for iron and zinc, where use of relatively large assessment factors leads to standards that are substantially below concentrations associated with impaired invertebrate assemblages in the field.

Deriving sediment quality guidelines from field-based species sensitivity distributions.

The determination of predicted no-effect concentrations (PNECs) and sediment quality guidelines (SQGs) of toxic chemicals in marine sediment is extremely important in ecological risk assessment. However, current methods of deriving sediment PNECs or threshold effect levels (TELs) are primarily based on laboratory ecotoxicity bioassays that may not be ecologically and environmentally relevant. This study explores the possibility of utilizing field data of benthic communities and contaminant loadings concurrently measured in sediment samples collected from the Norwegian continental shelf to derive SQGs. This unique dataset contains abundance data for ca. 2200 benthic species measured at over 4200 sampling stations, along with co-occurring concentration data for >25 chemical species. Using barium, cadmium, and total polycyclic aromatic hydrocarbons (PAHs) as examples, this paper describes a novel approach that makes use of the above data set for constructing field-based species sensitivity distributions (f-SSDs). Field-based SQGs are then derived based on the f-SSDs and HCx values [hazardous concentration for x% of species or the (100-x)% protection level] by the nonparametric bootstrap method. Our results for Cd and total PAHs indicate that there are some discrepancies between the SQGs currently in use in various countries and our field-data-derived SQGs. The field-data-derived criteria appear to be more environmentally relevant and realistic. Here, we suggest that the f-SSDs can be directly used as benchmarks for probabilistic risk assessment, while the field-data-derived SQGs can be used as site-specific guidelines or integrated into current SQGs.

Budding yeast Rad9 is an ATP-dependent Rad53 activating machine.

We find budding yeast Rad9 in two distinct, large, and soluble complexes in cell extracts. The larger (> or =850 kDa) complex, found in nondamaged cells, contains hypophosphorylated Rad9, whereas the smaller (560 kDa) complex, which forms after DNA damage, contains hyperphosphorylated Rad9 and Rad53. This smaller Rad9 complex is capable of catalyzing phosphorylation and release of active Rad53 kinase, a process requiring the kinase activity of Rad53. However, Mec1 and Tel1 are no longer required once the 560 kDa complex has been formed. We propose a model whereby Mec1/Tel1-dependent hyperphosphorylation of Rad9 results in formation of the smaller Rad9 complex and recruitment of Rad53. This complex then catalyzes activation of Rad53 by acting as a scaffold that brings Rad53 molecules into close proximity, facilitating Rad53 in trans autophosphorylation and subsequent release of activated Rad53.

Morphology of human neutrophils: a comparison of cryofixation, routine gluteraldehyde fixation, and the effects of dimethyl sulfoxide.

Cell shape and density are critical to the evaluation of neutrophil function and/or activation. Dimethyl sulfoxide-cryofixation-freeze-substitution processing (DCF) instantly preserves cell processes and ultrastructural elements with fewer artifacts than routine chemical fixation with glutaraldehyde and postfixation osmium tetroxide (GO). This study morphometrically examined density-separated neutrophils to assess differences in DCF and GO processing procedures and studied the effect of dimethyl sulfoxide followed by GO fixation (DGO) on morphology. Fifteen consecutive neutrophils were analyzed using computerized planimetry for differences in DCF v. GO treatments (n = 4) and DGO v. GO treatments (n = 4). Cryofixed and DGO-fixed cells were significantly rounder than GO cells which had a more irregular surface with membrane projections. The cell volume of GO cells was 27-30% smaller than in DCF or DGO processing, while the surface area was similar. The increased volume in DCF and DGO cells did not appear to be due to abnormal cell swelling, since membranes, nuclear envelope, and mitochondrial cristae were more intact than in GO cells. Preservation of mitochondria as well as endocytic caveolae with a subplasmalemmal coating was best in DCF samples, moderate in DGO, and poorest in GO. Morphometric data showed that the nuclear compartment was 22% smaller, while the cytoplasm (and its associated compartments) was 29% smaller in GO compared to DCF-processed neutrophils. This was consistent with the more dense cytoplasm in GO cells. Pretreatment of neutrophils with dimethyl sulfoxide (DMSO) resulted in volume preservation and improved the morphology of GO fixation. In summary, DCF appears to be an excellent method for preserving neutrophil membranes and cytoplasmic organelles (particularly mitochondria), and prevents a number of artifacts caused by routine GO fixation. Morphology can also be improved by using DMSO in conjunction with GO.

The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage.

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage. Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells. Treatment with various DNA damaging agents, i.e. UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein. All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation. Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage. Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

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.

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.

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.

Delay of vaccinia virus-induced apoptosis in nonpermissive Chinese hamster ovary cells by the cowpox virus CHOhr and adenovirus E1B 19K genes.

The infection of vaccinia virus in Chinese hamster ovary (CHO) cells produces a rapid shutdown in protein synthesis, and the infection is abortive (R.R. Drillien, D. Spehner, and A. Kirn, Virology 111:488-499, 1978; D.E. Hruby, D.L. Lynn, R. Condit, and J.R. Kates, J. Gen. Virol. 47:485-488, 1980). Cowpox virus, which can productively infect CHO cells, had previously been shown to contain a host range gene, CHOhr, which confers on vaccinia virus the ability to replicate in CHO cells (D. Spehner, S. Gillard, R. Drillien, and A. Kirn, J. Virol. 62:1297-1304, 1988). We found that CHO cells underwent apoptosis when infected with vaccinia virus. The expression of the CHOhr gene in vaccinia virus allowed for the expression of late virus genes. CHOhr also delayed or prevented vaccinia virus-induced apoptosis in CHO cells such that there was sufficient time for replication of the virus before the cell died. The E1B 19K gene from adenovirus also delayed vaccinia virus-induced apoptosis; however, there was no detectable expression of late virus genes. Furthermore, E1B 19K also delayed cell death in CHO cells which had been productively infected with vaccinia virus. This study identifies a new antiapoptotic gene from cowpox virus, CHOhr, for which the protein contains an ankyrin-like repeat and shows no significant homology to other proteins. This work also indicates that an antiapoptotic gene from one virus family can delay cell death in an infection of a virus from a different family.

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.