E. James Harner

Predicting Cancer Drug Response by Proteomic Profiling

Purpose: Accurate prediction of an individual patients drug response is an important prerequisite of personalized medicine. Recent pharmacogenomics research in chemosensitivity prediction has studied the gene-drug correlation based on transcriptional profiling. However, proteomic profiling will more directly solve the current functional and pharmacologic problems. We sought to determine whether proteomic signatures of untreated cells were sufficient for the prediction of drug response. Experimental Design: In this study, a machine learning model system was developed to classify cell line chemosensitivity exclusively based on proteomic profiling. Using reverse-phase protein lysate microarrays, protein expression levels were measured by 52 antibodies in a panel of 60 human cancer cell (NCI-60) lines. The model system combined several well-known algorithms, including random forests, Relief, and the nearest neighbor methods, to construct the protein expression–based chemosensitivity classifiers. The classifiers were designed to be independent of the tissue origin of the cells. Results: A total of 118 classifiers of the complete range of drug responses (sensitive, intermediate, and resistant) were generated for the evaluated anticancer drugs, one for each agent. The accuracy of chemosensitivity prediction of all the evaluated 118 agents was significantly higher (P < 0.02) than that of random prediction. Furthermore, our study found that the proteomic determinants for chemosensitivity of 5-fluorouracil were also potential diagnostic markers of colon cancer. Conclusions: The results showed that it was feasible to accurately predict chemosensitivity by proteomic approaches. This study provides a basis for the prediction of drug response based on protein markers in the untreated tumors.

EFFECTS OF THE EXXON VALDEZ OIL SPILL ON HABITAT USE BY BIRDS IN PRINCE WILLIAM SOUND, ALASKA

Oil spills may affect species through direct effects on population size and structure and direct and indirect (toxicological) effects on reproduction. Spill effects on the habitats these organisms occupy have received less attention, but they are no less important. For 2.5 yr following the Exxon Valdez oil spill in Prince William Sound, Alaska, we studied the use of oil-affected habitats by 42 species of marine-oriented birds. On 11 survey cruises, we surveyed bays that had received different levels of initial oiling. We related the abundance of individual species in the bays to the oiling gradient, using regression models that included habitat measures to control for variations among the sites in features other than oiling level. We defined a spill-induced impact as a statistically significant relationship between the abundance of a species and values along the oiling gradient, after accounting for the effects of variations in habitat features. We used among-year comparisons of regressions between oiling levels and abundance, controlled for season, to assess recovery. We concluded that recovery from a spill-induced impact had occurred when we no longer could detect a significant relationship between a species’ abundance and oiling levels. Overall, 23 (55%) of the 42 species exhibited no initial negative impacts on their use of oil-affected habitats. Of the 19 species that did exhibit negative impacts, 13 (68%) showed evidence of recovery within 2.5 yr (the final survey in 1991). Six species (Horned Grebe, Red-necked Grebe, Barrow’s Goldeneye, Bufflehead, Mew Gull, and Northwestern Crow) showed no clear evidence of recovery by our final survey. The proportion of species recorded on individual surveys that exhibited negative impacts at that time declined over the study, from 54% on the first survey after the spill in 1989 to 10% in late 1991. A principal components analysis revealed extensive ecological overlap between species that were negatively impacted in their use of oil-affected habitats and those that were not. The six species that had not recovered by late 1991 tended to be intertidal feeders and residents, but these traits also characterized some species that did not exhibit initial impacts and some species that subsequently recovered from impacts. We detected no obvious ecological differences between species that suffered spill impacts on habitat use and those that apparently were not affected, or between impacted species that later recovered in their use of habitats and species that had not yet recovered. These results indicate that the Exxon Valdez oil spill had clear initial negative impacts on habitat use by nearly half of the species examined, suggesting substantial initial effects on habitat suitability for these species. These impacts persisted for <2.5 yr for most affected species. This rate of recovery in habitat use parallels the rapid recovery (usually <2 yr) documented for other oil-affected communities (e.g., intertidal invertebrates, fishes, and birds) that have been studied in Alaska and elsewhere.

Multivariate measures of niche overlap using discriminant analysis

Abstract Two measures of multivariate niche overlap defined on p resource variables are presented. By measuring the niche overlap on the discriminant variable the multivariate problem is reduced to a univariate problem while preserving the relevant multivariate information. The niche overlap is then calculated by two different techniques. The first technique uses the MacArthur-Levins (Amer. Natur. 101, 377–385, 1967) measure for probabilities of joint occurrence, while the second computes the density overlap of two use curves. An illustration of the multivariate approach to actual field data is demonstrated.

Population-Based Molecular Prognosis of Breast Cancer by Transcriptional Profiling

Purpose: The purpose of this study is to predict breast cancer recurrence and metastases and to identify gene signatures indicative of clinicopathologic characteristics using gene expression patterns derived from cDNA microarray. Experimental Design: Expression profiles of 7,650 genes were investigated on an unselected group of 99 node-negative and node-positive breast cancer patients to identify prognostic gene signature of recurrence and metastases. The identified gene signature was validated on independent 78 patients with primary invasive carcinoma (T1/T2 and N0) and on 58 patients with locally advanced breast cancer (T3/T4 and/or N2). The gene predictors were identified using a combination of random forests and linear discriminant analysis function. Results: This study identified a new 28-gene signature that achieved highly accurate disease-free survival and overall survival (both at P < 0.001, time-dependent receiver operating characteristic analysis) in individual breast cancer patients. Patients categorized into high-risk, intermediate-risk, and low-risk groups had distinct disease-free survival (P < 0.005, Kaplan-Meier analysis, log-rank test) in three patient cohorts. A strong association (P < 0.05) was identified between risk groups and tumor size, tumor grade, estrogen receptor and progesterone receptor status, and HER2/neu overexpression in the studied cohorts. We also identified 14-gene predictors of nodal status and 9-gene predictors of tumor grade. Conclusions: This study has established a population-based approach to predicting breast cancer outcomes at the individual level exclusively based on gene expression patterns. The 28-gene recurrence signature has been validated as quantifying the probability of recurrence and metastases in patients with heterogeneous histology and disease stage.

THE LEGACY OF MERCURY CYCLING FROM MINING SOURCES IN AN AQUATIC ECOSYSTEM: FROM ORE TO ORGANISM

Clear Lake is the site of an abandoned mercury (Hg) mine (active intermittently from 1873 to 1957), now a U.S. Environmental Protection Agency Superfund Site. Mining activities, including bulldozing waste rock and tailings into the lake, resulted in approximately 100 Mg of Hg entering the lakes ecosystem. This series of papers represents the culmination of approximately 15 years of Hg-related studies on this ecosystem, following Hg from the ore body to the highest trophic levels. A series of physical, chemical, biological, and limnological studies elucidate how ongoing Hg loading to the lake is influenced by acid mine drainage and how wind-driven currents and baroclinic circulation patterns redistribute Hg throughout the lake. Methylmercury (MeHg) production in this system is controlled by both sulfate-reducing bacteria as well as newly identified iron-reducing bacteria. Sediment cores (dated with dichlorodiphenyldichlorethane [DDD], 210pb, and 14C) to approximately 250 cm depth (representing up to approximately 3000 years before present) elucidate a record of total Hg (TotHg) loading to the lake from natural sources and mining and demonstrate how MeHg remains stable at depth within the sediment column for decades to millenia. Core data also identify other stresses that have influenced the Clear Lake Basin especially over the past 150 years. Although Clear Lake is one of the most Hg-contaminated lakes in the world, biota do not exhibit MeHg concentrations as high as would be predicted based on the gross level of Hg loading. We compare Clear Lakes TotHg and MeHg concentrations with other sites worldwide and suggest several hypotheses to explain why this discrepancy exists. Based on our data, together with state and federal water and sediment quality criteria, we predict potential resulting environmental and human health effects and provide data that can assist remediation efforts.

IS CLEAR LAKE METHYLMERCURY DISTRIBUTION DECOUPLED FROM BULK MERCURY LOADING

Clear Lake is the site of the abandoned Sulphur Bank Mercury Mine, active periodically from 1873 to 1957, resulting in approximately 100 Mg of mercury (Hg) being deposited into the lakes ecosystem. Concentrations of total (primarily inorganic) Hg (TotHg) in Clear Lake are some of the highest reported worldwide for sediments (up to 4.4 x 10(5) ng/g [ppb dry mass]) and water (up to 4 x 10(-1) microg/L [= ppb]). However, the ratio of methylmercury (MeHg) to TotHg at Clear Lake indicates that the methylation process is mostly decoupled from bulk inorganic Hg loading, with Hg in lower trophic level biota significantly less than anticipated compared with other Hg-contaminated sites worldwide. This may be due to several factors, including: (1) reduced bioavailability of Hg derived from the mine (i.e., cinnabar, metacinnabar, and corderoite), (2) the alkaline nature of the lake water, (3) the shallow depth of the lake, which prevents stratification and subsequent methylation in a stratified hypolimnion, and (4) possible dilution of MeHg by a highly productive system. However, while bulk inorganic Hg loading to the lake may not contribute significantly to the bioaccumulation of Hg, acid mine drainage (AMD) from the mine likely promotes Hg methylation by sulfate-reducing and iron-reducing bacteria, making AMD a vehicle for the production of highly bioavailable Hg. If Clear Lake were deeper, less productive, or less alkaline, biota would likely contain much more MeHg than they do presently. Comparisons of MeHg:TotHg ratios in sediments, water, and biota from sites worldwide suggest that the highest production of MeHg may be found at sites influenced by chloralkali plants, followed by sites influenced by gold and silver mines, with the lowest production of MeHg observed at cinnabar and metacinnabar Hg mines. These data also suggest that the total maximum daily load (TMDL) process for Hg at Clear Lake, as currently implemented to reduce contamination in fishes for the protection of wildlife and humans, may be flawed because the metric used to implement Hg load reduction (i.e., TotHg) is not directly proportional to the critical form of Hg that is being bioaccumulated (i.e., MeHg).

MINE-DERIVED MERCURY: EFFECTS ON LOWER TROPHIC SPECIES IN CLEAR LAKE, CALIFORNIA

Considerable ecological research on mercury (Hg) has focused on higher trophic level species (e.g., fishes and birds), but less on lower trophic species. Clear Lake, site of the abandoned Sulphur Bank Mercury Mine, provides a unique opportunity to study a system influenced by mine-derived Hg. An exponentially decreasing gradient of total Hg (TotHg) away from the mine allowed us to evaluate Hg bioaccumulation in planktonic and benthic invertebrates and evaluate population- and community-level parameters that might be influenced by Hg. Studies from 1992-1998 demonstrated that TotHg in lower trophic species typically decreased exponentially away from the mine, similar to trends observed in water and sediments. However, a significant amount of invertebrate TotHg (approximately 60% for sediment-dwelling chironomid insect larvae) likely derives from Hg-laden particles in their guts. Spatially, whole-body methylmercury (MeHg) did not typically exhibit a significant decrease with increasing distance from the mine. Temporally, TotHg concentrations in plankton and chironomids did not exhibit any short-term (seasonal or annual) or long-term (multiyear) trends. Methylmercury, however, was elevated during late summer/fall in both plankton and chironomids, but it exhibited no long-term increase or decrease during this study. Although data from a 50-yr monitoring program for benthic chaoborid and chironomid larvae documented significant population fluctuations, they did not demonstrate population-level trends with respect to Hg concentrations. Littoral invertebrates also exhibited no detectable population- or community-level trends associated with the steep Hg gradient. Although sediment TotHg concentrations (1-1200 mg/kg dry mass) exceed sediment quality guidelines by up to 7000 times, it is notable that no population- or community-level effects were detected for benthic and planktonic taxa. In comparison with other sites worldwide, Clear Lakes lower trophic species typically have significantly higher TotHg concentrations, but comparable or lower MeHg concentrations, which may be responsible for the discrepancy between highly elevated TotHg concentrations and the general lack of observed population- or community-level effects. These data suggest that MeHg, as well as TotHg, should be used when establishing sediment quality guidelines. In addition, site-specific criteria should be established using the observed relationship between MeHg and observed ecological responses.

SPATIOTEMPORAL TRENDS IN FISH MERCURY FROM A MINE-DOMINATED ECOSYSTEM: CLEAR LAKE, CALIFORNIA

Clear Lake, California, USA, receives acid mine drainage and mercury (Hg) from the Sulphur Bank Mercury Mine, a U.S. Environmental Protection Agency (U.S. EPA) Superfund Site that was active intermittently from 1873 to 1957 and partially remediated in 1992. Mercury concentrations were analyzed primarily in four species of Clear Lake fishes: inland silversides (Menidia beryllina, planktivore), common carp (Cyprinus carpio, benthic scavenger/omnivore), channel catfish (Ictalurus punctatus, benthic omnivorous predator), and largemouth bass (Micropterus salmoides, piscivorous top predator). These data represent one of the largest fish Hg data sets for a single site, especially in California. Spatially, total Hg (TotHg) in silversides and bass declined with distance from the mine, indicating that the mine site represents a point source for Hg loading to Clear Lake. Temporally, fish Hg has not declined significantly over 12 years since mine site remediation. Mercury concentrations were variable throughout the study period, with no monotonic trends of increase or decrease, except those correlated with boom and bust cycles of an introduced fish, threadfin shad (Dorosoma petenense). However, stochastic events such as storms also influence juvenile largemouth bass Hg as evidenced during an acid mine drainage overflow event in 1995. Compared to other sites regionally and nationally, most fish in Clear Lake exhibit Hg concentrations similar to other Hg-contaminated sites, up to approximately 2.0 mg/kg wet mass (WM) TotHg in largemouth bass. However, even these elevated concentrations are less than would be anticipated from such high inorganic Hg loading to the lake. Mercury in some Clear Lake largemouth bass exceeded all human health fish consumption guidelines established over the past 25 years by the U.S. Food and Drug Administration (1.0 mg/kg WM), the National Academy of Sciences (0.5 mg/kg WM), and the U.S. EPA (0.3 mg/kg WM). Mercury in higher trophic level fishes exceeds ecotoxicological risk assessment estimates for concentrations that would be safe for wildlife, specifically the nonlisted Common Merganser and the recently delisted Bald Eagle. Fish populations of 11 out of 18 species surveyed exhibited a significant decrease in abundance with increasing proximity to the mine; this decrease is correlated with increasing water and sediment Hg. These trends may be related to Hg or other lake-wide gradients such as distribution of submerged aquatic vegetation.

MERCURY IN ABIOTIC MATRICES OF CLEAR LAKE, CALIFORNIA: HUMAN HEALTH AND ECOTOXICOLOGICAL IMPLICATIONS

Mercury (Hg) from Hg mining at Clear Lake, California, USA, has contaminated water and sediments for over 130 years and has the potential to affect human and environmental health. With total mercury (TotHg) concentrations up to 438 mg/kg (dry mass) in surficial sediments and up to 399 ng/L in lake water, Clear Lake is one of the most Hg-contaminated lakes worldwide. Particulate Hg in surface water near the mine ranges from 10,000 to 64,000 ng/g; TotHg declines exponentially with distance from the Sulphur Bank Mercury Mine. From 1992 to 1998, no significant long-term trends for TotHg or methylmercury (MeHg) in sediments or water were observed, but peaks of both TotHg and MeHg occurred following a 1995 flooding event. Sediments and water exhibit summer/fall maxima and winter/spring minima for MeHg, but not TotHg. Sediment TotHg has not declined significantly a decade after remediation in 1992. At the mine site, aqueous TotHg reached 374,000 ng/L in unfiltered groundwater. Pore water sulfate in sediments varies seasonally from 112 mg/L in summer/fall (when Hg methylation is highest) to 3300 mg/L in winter. While TotHg is exceptionally high in both sediments and water, MeHg is substantially lower than would be expected based on the bulk Hg loading to the lake and in comparison with other sites worldwide. Total mercury in Clear Lake water does not exceed the Safe Drinking Water Act criteria, but it sometimes greatly exceeds human health criteria established by the Great Lakes Initiative, U.S. Environmental Protection Agency water quality guidelines, and the California Toxics Rule criterion. Methylmercury concentrations exceed the Great Lakes Initiative criterion for MeHg in water at some sites only during summer/fall. Relative to ecological health, Clear Lake sediments greatly exceed the National Oceanic and Atmospheric Administrations benthic fauna Sediment Quality Guidelines for toxic effects, as well as the more concensus-based Threshold Effects Concentration criteria. Based on these criteria, Hg-contaminated sediments and water from Clear Lake are predicted to have some lethal and sublethal effects on specific resident aquatic species. However, based on unique physical and chemical characteristics of the Clear Lake environment, MeHg toxicity may be significantly less than anticipated from the large inorganic Hg loading.

There Is No Need To Be Normal: Generalized Linear Models of Natural Variation

Both ecological field studies and attempts to extrapolate from laboratory experiments to natural populations generally encounter the high degree of natural variability and chaotic behavior that typify natural ecosystems. Regardless of this variability and non-normal distribution, most statistical models of natural systems use normal error which assumes independence between the variance and mean. However, environmental data are often random or clustered and are better described by probability distributions which have more realistic variance to mean relationships. Until recently statistical software packages modeled only with normal error and researchers had to assume approximate normality on the original or transformed scale of measurement and had to live with the consequences of often incorrectly assuming independence between the variance and mean. Recent developments in statistical software allow researchers to use generalized linear models (GLMs) and analysis can now proceed with probability distributio...