Transformation of mercury at the bottom of the Arctic food web: an overlooked puzzle in the mercury exposure narrative.

We show 2008 seasonal trends of total and monomethyl mercury (THg and MeHg, respectively) in herbivorous (Calanus hyperboreus) and predatory (Chaetognaths, Paraeuchaeta glacialis, and Themisto abyssorum) zooplankton species from the Canadian High Arctic (Amundsen Gulf and the Canadian Beaufort Sea) in relation to ambient seawater and diet. It has recently been postulated that the Arctic marine environment may be exceptionally vulnerable to toxic MeHg contamination through postdepositional processes leading to mercury transformation and methylation. Here, we show that C. hyperboreus plays a hitherto unrecognized central role in mercury transformation while, itself, not manifesting inordinately high levels of THg compared to its prey (pelagic particulate organic matter (POM)). Calanus hyperboreus shifts Hg from mainly inorganic forms in pelagic POM (>99.5%) or ambient seawater (>90%) to primarily organic forms (>50%) in their tissue. We calculate that annual dietary intake of MeHg could supply only ∼30% of the MeHg body burden in C. hyperboreus and, thus, transformation within the species, perhaps mediated by gut microbial communities, or bioconcentration from ambient seawater likely play overriding roles. Seasonal THg trends in C. hyperboreus are variable and directly controlled by species-specific physiology, e.g., egg laying and grazing. Zooplankton that prey on species such as C. hyperboreus provide a further biomagnification of MeHg and reflect seasonal trends observed in their prey.

Importance of Arctic zooplankton seasonal migrations for α-hexachlorocyclohexane bioaccumulation dynamics.

Like most zooplankton, Calanus hyperboreus undergoes seasonal migration spending late spring and summer grazing at the surface and the rest of the year in diapause at depth. As a result, in the Arctic Ocean this copepod resides for part of the year in the hexachlorocyclohexane (HCH) enriched surface water and for part of the year at depth where HCH undergoes significant microbial degradation resulting in far lower concentrations (~3 times for α-HCH). We collected C. hyperboreus from summer and winter from the Amundsen Gulf and measured their α-HCH concentrations, enantiomeric compositions, and bioaccumulation factors (BAFs) to investigate how this copepod responds to the change in exposure to α-HCH. C. hyperboreus collected in winter were also cultured for 5 weeks under surface water conditions without feeding to investigate bioconcentration dynamics following spring ascent. Concentration of α-HCH was 2-3 times higher in individuals from the summer than those from the winter. Log BAF from the summer (feeding period) does not exceed log BCF (bioconcentration factor) from the culturing experiment (no feeding) suggesting that α-HCH concentration in C. hyperboreus is maintained through equilibration rather than feeding. After the spring ascent from deep waters, C. hyperboreus approach equilibrium partitioning with the higher surface water concentrations of α-HCH within 3-4 weeks with about 60% of bioconcentration taking place in the first week. The C. hyperboreus α-HCH chiral signature also reflects ambient seawater and can therefore be used as a determinant of residence depth. Even though a single cycle of seasonal migration does not result in a significant redistribution of α-HCH in the water column, this process could have a significant cumulative effect over longer time scales with particular local importance where the zooplankton biomass is high and the ocean depth is great enough to provide substantial vertical concentration gradients.

Western Canadian Arctic ringed seal organic contaminant trends in relation to sea ice break-up.

The association between changing sea ice conditions and contaminant exposure to Arctic animals interests Inuvialuit harvesters, communities, and researchers. We examined organochlorine contaminant (OC) concentrations in the blubber of 90 male adult ringed seals (Phoca hispida) sampled from the subsistence harvest in Ulukhaktok (formerly Holman), NT, Canada, just prior to break-up of the sea ice (1993-2008). OC blubber concentrations were assessed with respect to year and sea ice break-up date. HCB and age- and blubber-adjusted concentrations of p,p'-DDT and ΣCHB (chlorobornane) significantly decreased over the study period. With respect to the timing of the spring break-up, highly lipophlic OCs, such as p,p'-DDE and PCB 153, were higher during years of early ice clearing (at least 12 days earlier than the mean annual break-up date), whereas no trends were observed for α, ß, and γ isomers of HCH, trans- and cis-chlordane, oxychlordane, or ΣCHB. The higher contaminant concentrations found in earlier break-up years is likely due to earlier and/or increased foraging opportunities. This situation also has potential for enhancing bioaccumulation and biomagnification of contaminants over the long-term if projected changes continue to result in lighter and earlier ice conditions.

Mechanisms and implications of α-HCH enrichment in melt pond water on Arctic sea ice.

During the summer of 2009, we sampled 14 partially refrozen melt ponds and the top 1 m of old ice in the pond vicinity for α-hexachlorocyclohexane (α-HCH) concentrations and enantiomer fractions (EFs) in the Beaufort Sea. α-HCH concentrations were 3 - 9 times higher in melt ponds than in the old ice. We identify two routes of α-HCH enrichment in the ice over the summer. First, atmospheric gas deposition results in an increase of α-HCH concentration from 0.07 ± 0.02 ng/L (old ice) to 0.34 ± 0.08 ng/L, or ~20% less than the atmosphere-water equilibrium partitioning concentration (0.43 ng/L). Second, late-season ice permeability and/or complete ice thawing at the bottom of ponds permit α-HCH rich seawater (~0.88 ng/L) to replenish pond water, bringing concentrations up to 0.75 ± 0.06 ng/L. α-HCH pond enrichment may lead to substantial concentration patchiness in old ice floes, and changed exposures to biota as the surface meltwater eventually reaches the ocean through various drainage mechanisms. Melt pond concentrations of α-HCH were relatively high prior to the late 1980-s, with a Melt pond Enrichment Factor >1 (MEF; a ratio of concentration in surface meltwater to surface seawater), providing for the potential of increased biological exposures.

α- and γ-Hexachlorocyclohexane measurements in the brine fraction of sea ice in the Canadian High Arctic using a sump-hole technique.

We used holes augered partially into first-year sea ice (sumps) to determine α- and γ-HCH concentrations in sea-ice brine. The overwintering of the CCGS Amundsen in the Canadian western Arctic, as part of the Circumpolar Flaw Lead (CFL) System Study, provided the circumstances to allow brine to accumulate in sumps sufficiently to test the methodology. We show, for the first time, that as much as 50% of total HCHs in seawater can become entrapped within the ice crystal matrix. On average, in the winter first-year sea ice HCH brine concentrations reached 4.013 ± 0.307 ng/L and 0.423 ± 0.013 ng/L for the α- and γ-isomer, respectively. In the spring, HCHs decreased gradually with time, with increasing brine volume fraction and decreasing brine salinity. These decreasing concentrations could be accounted for by both the dilution with the ice crystal matrix and under-ice seawater. We propose that the former process plays a more significant role considering brine volume fractions calculated in this study were below 20%. Levels of HCHs in the brine exceed under-ice water concentrations by approximately a factor of 3, a circumstance suggesting that the brine ecosystem has been, and continues to be, the most exposed to HCHs.

Temporal trends of organochlorine contaminants in burbot and lake trout from three selected Yukon lakes.

Historical studies have demonstrated that organochlorine (OC) concentrations in top predators can vary considerably from lake to lake within a small geographic region but temporal trends of these contaminants have rarely been monitored in a sub-Arctic area for a long period of time. This study examined OC concentrations, including chlordane (CHL), DDT, hexachlorocyclohexane (HCH), toxaphene (CHB), PCB and chlorinated benzenes (CBz) in lake trout and burbot, from three Yukon lakes (Laberge, Kusawa, Quiet), over a span of 11 years (1992-2003). Temporal and spatial differences continue to exist in the OC concentrations of burbot and lake trout between these lakes. There is strong evidence that these contaminants are declining at various rates in lake trout (Salveninus namaycush) in Laberge, Kusawa and Quiet Lakes. For example, SigmaDDT concentrations have decreased 39%, 85% and 84% in Kusawa, Quiet and Laberge Lakes, respectively. Conversely, no consistent trends were observed in OC concentrations for burbot (Lota lota). For example, there is no evidence of a decline in toxaphene concentrations of Kusawa burbot yet a 58% decrease was observed in Laberge samples. Increases were also observed in the SigmaHCH levels of Kusawa Lake burbot, as well as increases in all OC groups (except SigmaHCH) for the Quiet Lake burbot samples. Decreases in burbot were evident in SigmaHCH and SigmaCHB for Lake Laberge fish and in SigmaCHL for Kusawa Lake samples. Spatial variations in OC levels are quite evident as Lake Laberge trout and burbot continued to maintain the highest levels over the eleven-year period from 1992 to 2003 followed by Kusawa Lake and then Quiet Lake. These differences were related to a variety of factors especially the species morphological characteristics such as log age, log weights and fish lipid content. A decreasing trend in Quiet and Laberge Lake trout lipid content, coupled with fluctuating condition factors and increases in body masses, suggest biotic changes may be occurring within the food webs due to fish population variations related to the cessation of commercial fishing or potentially an increase in lake plankton productivity related to annual climate variation. It is suspected that biotic factors rather than atmospheric inputs are the primary factors affecting the contaminant concentrations in lake trout and burbot in the study lakes.

Spatial trends and factors affecting variation of organochlorine contaminants levels in Canadian Arctic beluga (Delphinapterus leucas).

Organochlorine pesticides and PCBs were analysed in blubber from beluga (Delphinapterus leucas), or white whales, collected at 15 sites in the Canadian Arctic between 1993 and 2001. The objective of the study was to define and interpret the spatial trends of major organic contaminants in northern beluga in terms of sources and transport pathways, and the biological factors influencing accumulation. When compared on a lipid weight basis, the concentrations of beta-HCH, cis-CHL and SigmaCHL, cis-nonachlor, heptachlor epoxide and p,p'-DDT were significantly higher in males than females at all five sites in the eastern Arctic where the two sexes were harvested. The differences were attributed to losses from the females during fetal development and lactation as reported in previous studies. Major compounds increased with age in males at most sites, however the lack of a significant increase with age at some sites was in part due to high organochlorine concentrations in young year classes (2-5 years), particularly at eastern sites such as Iqaluit and Pangnirtung. Lower concentrations of SigmaHCH and SigmaDDT compounds in young males in 2001 relative to 1995 at Hendrickson Island could be due to declining levels in the environment, changes in the diet, or differences in organochlorine loads transferred from the female after birth. Age-corrected least square mean concentrations in males showed significantly higher levels of many compounds, such as p,p'-DDE and SigmaCHB, at south Baffin Island sites than those in the west. Two notable exceptions were HCBz and beta-HCH which were higher in the west. Methoxyclor was detected in males at Sanikiluaq (58 ng g-1) and in both sexes at Kimmirut, but at no other sites. Principal component analysis grouped the 16 sites into five major groupings based on the similarity of normalised organochlorine pesticide and PCB levels. Sites from the western Arctic were grouped by higher proportions of HCBz, beta-HCH and gamma-HCH and higher chlorinated PCBs. Endosulfan and alpha-HCH comprised a larger proportion of total organochlorine residues in the northern Hudson Bay sites, while methoxychlor, chlordane compounds and octachlorobiphenyls were enriched at Sanikiluaq in eastern Hudson Bay. The analysis showed that the relative amounts of several key compounds are similar in the beluga stocks over large spatial areas (i.e. eastern versus western sites), however, some stocks have distinct fingerprints which can be used to differentiate them from adjacent stocks. Ratios of major HCH isomers largely corresponded with air and surface water measurements conducted during the 1990s, but low alpha-/beta- and alpha-/gamma-HCH ratios in all three western Arctic collections indicate rapid losses of the alpha-isomer from the food web, proportionately higher beta- and gamma-isomers in the Beaufort Sea, or a combination of the two processes. Chlordane residue patterns generally correspond to those from previous studies, however, interpretation of spatial trends are difficult due to the aging of the probable sources in the south, possible atmospheric input from new sources and complex transport pathways.

Temporal and spatial variabilities of atmospheric polychlorinated biphenyls (PCBs), organochlorine (OC) pesticides and polycyclic aromatic hydrocarbons (PAHs) in the Canadian Arctic: results from a decade of monitoring.

The Northern Contaminants Program (NCP) baseline monitoring project was established in 1992 to monitor for persistent organic pollutants (POPs) in Arctic air. Under this project, weekly samples of air were collected at four Canadian and two Russian arctic sites, namely Alert, Nunavut; Tagish, Yukon; Little Fox Lake, Yukon; Kinngait, Nunavut; Dunai Island, Russia and Amderma, Russia. Selected POPs, including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and organochlorine (OC) pesticides, were analyzed in both the gas and particulate phases. This paper summarizes results obtained from this project in the past 5 years. Temporal trends were developed for atmospheric PCBs and OCs observed at Alert using a digital filtration (DF) technique. It was found that trends developed with 5 years of data (1993-1997) did not differ significantly from those determined with 7 years of data (1993-1999). This implies that with the DF technique, long-term trends can still be developed with less than 10 years of data. An acceleration in decline of OC and PCB air concentrations was noted in 1999 for some compounds, although the reason is unknown. Monitoring efforts must continue to assess the effect of this decline on the long-term trends of POPs in the Canadian Arctic. Occasional high trans-/cis-chlordane ratios and heptachlor air concentrations measured at Alert between 1995 and 1997 suggests sporadic fresh usage of chlordane-based pesticides. However, significant decreasing trends of chlordanes along with their chemical signatures has provided evidence that emission of old soil residues is replacing new usage as an important source to the atmosphere. Measurements of OC air concentrations conducted at Kinngait in 1994-1995 and 2000-2001 indicated faster OC removal at this location than at Alert. This may be attributed to the proximity of Kinngait to temperate regions where both biotic and abiotic degradation rates are faster. The PAH concentrations observed at Alert mimic those at mid-latitudes and are consistent with long-range transport to the Arctic, particularly for the lighter PAHs. A decline in particulate PAH was observed, similar to atmospheric sulphate aerosol and can be attributed to the collapse of industrial activity in the former Soviet Union between 1991 and 1995. Spatial comparisons of OC seasonality at Alert, Tagish, Dunai and Kinngait show elevated air concentrations of some compounds in spring. However, elevated spring concentrations were observed for different compounds at different sites. Potential causes are discussed. Further investigation in the atmospheric flow pattern in spring which is responsible for the transport of POPs into the Arctic is required. OC and PCB air concentrations at Alert were found to be influenced by two climate variation patterns, the North Atlantic Oscillation (NAO) and the Pacific North American (PNA) pattern. Planetary atmospheric patterns must be taken into account in the global prediction and modelling of POPs in the future.

Modern and historical fluxes of halogenated organic contaminants to a lake in the Canadian arctic, as determined from annually laminated sediment cores.

Two annually laminated cores collected from Lake DV09 on Devon Island in May 1999 were dated using 210Pb and 137Cs, and analyzed for a variety of halogenated organic contaminants (HOCs), including polychlorinated biphenyls (PCBs), organochlorine pesticides, short-chain polychlorinated n-alkanes (sPCAs), polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), and polybrominated diphenyl ethers (PBDEs). Dry weight HOC concentrations in Lake DV09 sediments were generally similar to other remote Arctic lakes. Maximum HOC fluxes often agreed well with production maxima, although many compound groups exhibited maxima at or near the sediment surface, much later than peak production. The lower than expected HOC concentrations in older sediment slices may be due to anaerobic degradation and possibly to dilution resulting from a temporary increase in sedimentation rate observed between the mid-1960s and 1970s. Indeed, temporal trends were more readily apparent for those compound classes when anaerobic metabolites were also analyzed, such as for DDT and toxaphene. However, it is postulated here for the first time that the maximum or increasing HOC surface fluxes observed for many of the major compound classes in DV09 sediments may be influenced by climate variation and the resulting increase in algal primary productivity which could drive an increasing rate of HOC scavenging from the water column. Both the fraction (F(TC)) and enantiomer fraction (EF) of trans-chlordane (TC) decreased significantly between 1957 and 1997, suggesting that recent inputs to the lake are from weathered chlordane sources. PCDD/Fs showed a change in sources from pentachlorophenol (PeCP) in the 1950s and 1960s to combustion sources into the 1990s. Improvements in combustion technology may be responsible for the reducing the proportion of TCDF relative to OCDD in the most recent slice.

Concentrations of mercury in tissues of beluga whales (Delphinapterus leucas) from several communities in the Canadian Arctic from 1981 to 2002.

Beluga whales have been hunted for food by Native People in the Canadian Arctic since prehistoric time. Here we report the results of analyses of total mercury in samples of liver, kidney, muscle and muktuk from collections over the period 1981-2002. We compare these results with human consumption guidelines and examine temporal and geographic variation. Liver has been analyzed more frequently than other organs and it has been used as the indicator organ. Mercury accumulates in the liver of the whales over time so that the whale ages are usually linked statistically to their levels of mercury in liver. Virtually all the samples of 566 animals analyzed contained mercury in liver at concentrations higher than the Canadian consumption guideline of 0.5 microg g-1 (wet weight) for fish. (There is no regulatory guideline for concentrations in marine mammals in Canada.) Samples from locations in the Mackenzie Delta in the western Canadian Arctic and from Pangnirtung in the eastern Canadian Arctic were obtained more often than from other location and these offered the best chances to determine whether levels have changed over time. Statistical outlier points were removed and the regressions of (ln) mercury in liver on age were used to calculate the level of mercury in whales of age 13.1 years in order to compare age-adjusted levels at different locations. These age-adjusted levels and also the slopes of regressions suggested that levels have increased in the Mackenzie Delta over the sampling period although not in a simple linear fashion. Other locations had fewer collections, generally spread over fewer years. Some of them indicated differences between sampling times but we could not establish whether these differences were simply temporal variation or whether they were segments of a consistent trend. For example, the levels in whales from Arviat were considerably higher in 1999 than in 1984 but we have only two samples. Similarly, samples from Iqaluit in 1994 exceeded considerably those in 1993 and the interval seems too short to reflect any regional temporal trend and more likely represent an extreme case of year-to-year variation. Previous analyses of data from geographically distinct groups had suggested that whales in the western Canadian Arctic had higher levels of mercury than those from the eastern Canadian Arctic. The present analysis suggests that such regional differences have diminished and are no longer statistically significant. No site has indicated significant decreases in more recent samples. The levels of total mercury in the most analyzed organs fell in the order of liver (highest levels), kidney, muscle and muktuk (lowest level). While muktuk had the lowest level of the organs most frequently analyzed, it is the preferred food item from these whales and it still exceeded the consumption guideline in most instances.

Persistent organic pollutants and mercury in marine biota of the Canadian Arctic: an overview of spatial and temporal trends.

This review summarizes and synthesizes the significant amount of data which was generated on mercury (Hg) and persistent organic pollutants (POPs) in Canadian Arctic marine biota since the first Canadian Arctic Contaminants Assessment Report (CACAR) was published in 1997. This recent body of work has led to a better understanding of the current levels and spatial and temporal trends of contaminants in biota, including the marine food species that northern peoples traditionally consume. Compared to other circumpolar countries, concentrations of many organochlorines (OCs) in Canadian Arctic marine biota are generally lower than in the European Arctic and eastern Greenland but are higher than in Alaska, whereas Hg concentrations are substantially higher in Canada than elsewhere. Spatial coverage of OCs in ringed seals, beluga and seabirds remains a strength of the Arctic contaminant data set for Canada. Concentrations of OCs in marine mammals and seabirds remain fairly consistent across the Canadian Arctic although subtle differences from west to east and south to north are found in the proportions of various chemicals. The most significant development since 1997 is improvement in the temporal trend data sets, thanks to the use of archived tissue samples from the 1970s and 1980s, long-term studies using archeological material, as well as the continuation of sampling. These data cover a range of species and chemicals and also include retrospective studies on new chemicals such as polybrominated diphenyl ethers. There is solid evidence in a few species (beluga, polar bear, blue mussels) that Hg at some locations has significantly increased from pre-industrial times to the present; however, the temporal trends of Hg over the past 20-30 years are inconsistent. Some animal populations exhibited significant increases in Hg whereas others did not. Therefore, it is currently not possible to determine if anthropogenic Hg is generally increasing in Canadian Arctic biota. It is also not yet possible to evaluate whether the recent Hg increases observed in some biota may be due solely to increased anthropogenic inputs or are in part the product of environmental change, e.g., climate warming. Concentrations of most "legacy" OCs (PCBs, DDT, etc.) significantly declined in Canadian Arctic biota from the 1970s to the late 1990s, and today are generally less than half the levels of the 1970s, particularly in seabirds and ringed seals. Chlorobenzenes and endosulfan were among the few OCs to show increases during this period while summation operatorHCH remained relatively constant in most species. A suite of new-use chemicals previously unreported in Arctic biota (e.g., polybrominated diphenyl ethers (PBDEs), short chain chlorinated paraffins (SCCPs), polychlorinated naphthalenes (PCNs), perfluoro-octane sulfonic acid (PFOS) and perfluorocarboxylic acids (PFCAs)) has recently been found, but there is insufficient information to assess species differences, spatial patterns or food web dynamics for these compounds. Concentrations of these new chemicals are generally lower than legacy OCs, but there is concern because some are rapidly increasing in concentration (e.g., PBDEs), while others such as PFOS have unique toxicological properties, and some were not expected to be found in the Arctic because of their supposedly low potential for long-range transport. Continuing temporal monitoring of POPs and Hg in a variety of marine biota must be a priority.

A history of total mercury in edible muscle of fish from lakes in northern Canada.

Subsistence fishing has been an important source of food for Native People in northern Canada since prehistoric time. Measurements of the levels of mercury in edible muscle of northern fish have been undertaken for over three decades in efforts to evaluate the risks of consuming northern fish. This report summarizes the data obtained from 7974 fish of 25 species from sites distributed from the Yukon to Labrador. The most abundant species were lake trout, lake whitefish, arctic char, walleye, northern pike and burbot. The question being asked was essentially "Are the fish safe to eat?" The results were used to support decisions on fishing and consumption of fish. They were sorted in several ways, into concentration ranges corresponding to human consumption guidelines, into political jurisdictions and into types of bedrock geology. Overall walleye, northern pike and lake trout, usually exceeded the subsistence consumption guideline of 0.2 microg g-1 total mercury and often exceeded the higher guideline of 0.5 microg g-1 total mercury for commercial sales of fish. Mercury in burbot, another facultative predator, was often lower but several still exceeding a guideline. Arctic char collections were mostly from anadromous populations and these had very low levels of mercury, presumably reflecting marine food sources. Lake whitefish were among the cleanest fish examined with 69 of 81 collections falling in the lowest range. Most collections were from sites in sedimentary rock. However a few sites were in metamorphic, intrusive or volcanic rocks and these, taken together, tended to have a higher proportion of sites in the higher ranges of mercury. These results indicate a widespread problem with mercury in subsistence fisheries for predator species of fish with the problem being most problematic for Nunavut.

Distant drivers or local signals: where do mercury trends in western Arctic belugas originate?

Temporal trends of contaminants are monitored in Arctic higher trophic level species to inform us on the fate, transport and risk of contaminants as well as advise on global emissions. However, monitoring mercury (Hg) trends in species such as belugas challenge us, as their tissue concentrations reflect complex interactions among Hg deposition and methylation, whale physiology, dietary exposure and foraging patterns. The Beaufort Sea beluga population showed significant increases in Hg during the 1990 s; since that time an additional 10 years of data have been collected. During this time of data collection, changes in the Arctic have affected many processes that underlie the Hg cycle. Here, we examine Hg in beluga tissues and investigate factors that could contribute to the observed trends after removing the effect of age and size on Hg concentrations and dietary factors. Finally, we examine available indicators of climate variability (Arctic Oscillation (AO), the Pacific Decadal Oscillation (PDO) and sea-ice minimum (SIM) concentration) to evaluate their potential to explain beluga Hg trends. Results reveal a decline in Hg concentrations from 2002 to 2012 in the liver of older whales and the muscle of large whales. The temporal increases in Hg in the 1990 s followed by recent declines do not follow trends in Hg emission, and are not easily explained by diet markers highlighting the complexity of feeding, food web dynamics and Hg uptake. Among the regional-scale climate variables the PDO exhibited the most significant relationship with beluga Hg at an eight year lag time. This distant signal points us to consider beluga winter feeding areas. Given that changes in climate will impact ecosystems; it is plausible that these climate variables are important in explaining beluga Hg trends. Such relationships require further investigation of the multiple connections between climate variables and beluga Hg.

Elevation of transforming growth factor alpha in cat aqueous humor after corneal endothelial injury.

PURPOSE: To determine if a scrape injury to cat corneal endothelial cells increases the level of mitogenic proteins such as transforming growth factor alpha (TGF alpha) in aqueous humor. METHODS: Aqueous humor of cats was collected at 0, 2, 6, and 24 hours after wounding the endothelium by contact with a cannula tip. Aqueous humor samples collected from sham-wounded cats served as controls. Aqueous humor samples were analyzed for levels of protein, for mitogenic activity using incorporation of tritiated thymidine by cultures of bovine corneal endothelial cells, and for immunoreactive TGF alpha protein using a specific radioimmunoassay. RESULTS: The average protein level in aqueous humor obtained before wounding was low (0.5 mg/ml), increased 26-fold at 2 hours after injury (13 mg/ml), then progressively decreased at 6 hours (8 mg/ml) and 24 hours (2 mg/ml). Levels of mitogenic activity of aqueous humor samples collected 2, 6, and 24 hours after wounding were 2-fold, 2.5-fold, and 0.6-fold higher, respectively, compared to the level of mitogenic activity measured in aqueous humor collected before wounding (0 hours) or in aqueous humor collected from sham-wounded eyes. TGF alpha concentration in aqueous humor collected before endothelial wounding was low (6.8 ng/ml), increased 14-fold 2 hours after wounding (97.4 ng/ml), then progressively decreased at 6 hours (63.3 ng/ml) and 24 hours (35.5 ng/ml) after wounding. TGF alpha concentrations in aqueous humor collected from sham-wounded eyes at 2 hours (9.5 ng/ml) and 6 hours (5.3 ng/ml) were not significantly different from prewound levels. Detergent extracts of bovine corneal endothelial cells contained substantial levels of TGF alpha immunoreactive protein (20 ng/mg protein). CONCLUSIONS: Wounding of cat endothelium causes a rapid increase in mitogenic proteins in aqueous humor including TGF alpha, which may act by an autocrine mechanism to stimulate endothelial wound healing.

Treatment of alkali-injured rabbit corneas with a synthetic inhibitor of matrix metalloproteinases.

Healing of corneal alkali injuries remains a severe clinical challenge. The authors evaluated the effect of a new synthetic inhibitor of matrix metalloproteinases (GM6001 or N-[2(R)-2-(hydroxamido carbonylmethyl)-4-methylpentanoyl]-L-tryptophane methylamide) on preventing ulceration of rabbit corneas after alkali injury. Topical treatment of corneas with severe alkali injuries with 400 micrograms/ml or 40 micrograms/ml GM6001 alone prevented ulceration for 28 days, although 8 of 10 corneas treated with vehicle perforated. Corneas treated with 4 micrograms/ml GM6001 had midstromal depth ulcers. Corneas treated with 400 micrograms/ml of GM6001 contained very few inflammatory cells and had significantly reduced vessel ingrowth compared with vehicle-treated corneas. Epithelial regeneration after moderate alkali injuries also was investigated. Persistent epithelial defects developed 4 days after moderate alkali injury in rabbit corneas treated with vehicle and progressively increased to an average of 20% of the original 6 mm diameter wound by 27 days after moderate alkali injury. By contrast, epithelial regeneration was complete and persisted for 21 days for corneas treated with a formulation containing GM6001 (400 micrograms/ml), epidermal growth factor (10 micrograms/ml), fibronectin (500 micrograms/ml), and aprotinin (400 micrograms/ml). Sporadic punctate staining developed in 20% of the corneas treated with the combination of agents between days 21-28 after moderate alkali injury. These results demonstrate that topical application of GM6001 prevented corneal ulceration after severe alkali injury and that a combination containing GM6001, epidermal growth factor, fibronectin, and aprotinin promoted stable regeneration of corneal epithelium after moderate alkali injury.

Inhibition of pseudomonal ulceration in rabbit corneas by a synthetic matrix metalloproteinase inhibitor.

PURPOSE: To evaluate the effect of the synthetic matrix metalloproteinase inhibitor, Galardin, on proteases produced by Pseudomonas aeruginosa (PA) and on a rabbit model of Pseudomonas keratitis. METHODS: Protease activities of culture broths from Pseudomonas strains PA-28 and W-186 were characterized in vitro by gelatin zymography and by digestion of Azocasein in the presence and absence of Galardin and the serine protease inhibitor, aprotinin. In a noninfectious in vivo experiment, sterile PA culture broth from W-186 was injected intrastromally into rabbit corneas that were treated topically with Galardin or vehicle, then evaluated clinically and histologically. In an infectious in vivo experiment, rabbit corneas were injected with washed PA-28, then treated topically with Galardin or vehicle and clinically scored. RESULTS: Gelatin zymography of culture broth from W-186 and PA-28 detected two proteases that were both inhibited by Galardin. Galardin reduced the digestion of Azocasein by both PA culture broths by 99%, whereas aprotinin did not significantly reduce the protease activity of PA-28 conditioned broth. Intrastromal injection of sterile W-186 culture broth caused rapid corneal destruction that was prevented by topical treatment with Galardin. Intrastromal injection of washed PA-28 bacteria resulted in progressive corneal melting that was significantly (P < 0.005) delayed, but ultimately not prevented, by topical treatment with Galardin. CONCLUSIONS: Pseudomonal protease activity in culture broth consisted predominantly of metalloproteinases and were effectively inhibited by Galardin in vitro. Topical treatment with Galardin prevented destruction of rabbit corneas by bacterial products present in culture broth, and it delayed corneal destruction after injection of PA bacteria. Galardin may be a useful adjuvant when corneal destruction proceeds despite prompt antibiotic treatment.

Enhanced healing of cat corneal endothelial wounds by epidermal growth factor.

PURPOSE: The authors investigated whether healing of cat corneal endothelial wounds could be enhanced in vivo by human epidermal growth factor (EGF). METHODS: EGF was administered in sodium hyaluronate to the anterior chamber of cats after an endothelial touch injury. Control contralateral eyes received sodium hyaluronate alone. At selected times after injury, the corneas were evaluated for thickness, the rate of endothelial wound closure, the endothelial cell density, any variation in cell size, the percentage of hexagonal cells, and endothelial cell mitosis. RESULTS: Two days after injury, endothelial wounds of eyes treated with EGF had healed an average of 65 +/- 4% of the initial 38.5 mm2 wound area; paired control eyes had healed an average of 59 +/- 4% (P < 0.05). Both EGF-treated and control wounds had resurfaced over 90% of the initial wound area on day 4 after injury, and the wounds were completely resurfaced by 7 and 14 days after injury in both treatment groups. On days 4 and 7 after injury, the EGF-treated corneas were 5% and 8% thicker (835 versus 796 microns and 786 versus 728 microns, respectively) than the paired control corneas (P < 0.03). On days 10 and 14 after injury, both EGF-treated and control corneas were 19% and 12% thicker, respectively, than prewound the corneal thickness (621 microns). Seven days after injury, the corneas treated with EGF had an average of 76 +/- 28% more (P < 0.05) endothelial cell nuclei labeled with tritiated thymidine compared with that of the paired control eyes (2472 versus 1543 labeled nuclei). Fourteen days after injury, the central endothelial cell density of EGF-treated corneas was an average of 38 +/- 11% higher than that of the paired control eyes (P < 0.01, 1708 versus 1235 cells/mm2). The percentage of hexagonal cells in the wound area was an average of 14 +/- 4% higher (P < 0.01) than that of the paired control eyes (82% versus 69%), and the coefficient of variation of the cell size for EGF-treated corneas was an average of 31% (P < 0.05) smaller than that of the paired control corneas (0.21 versus 0.29 [standard deviation]/mean cell size). CONCLUSIONS: A single intraocular application of EGF formulated in sodium hyaluronate after an endothelial cell injury significantly enhanced multiple parameters that are closely related to improved endothelial cell regeneration.

Teaching ophthalmology to primary care physicians. The Association of University Professors of Ophthalmology Education Committee.

The evolution of our health care system toward managed care has placed primary care "gatekeeper" physicians in a position where they must examine and treat patients with diseases that are ordinarily treated by medical and surgical subspecialists. Many of these physicians may be poorly prepared to accept this new responsibility. The Association of University Professors of Ophthalmology (AUPO) has published a Policy Statement on Medical Student Education that suggests the minimum level of competence expected of general physicians when dealing with ophthalmologic problems. A recent survey of directors of residency programs in family practice, internal medicine, and pediatrics revealed that the training of primary care physicians (as opposed to academicians or subspecialists) has become a nearly universal programmatic goal. Most program directs believe that less than half of their residents meet the AUPO standard at the completion of medical school, and a significant minority do not meet the AUPO standard at the completion of their residency. An overwhelming majority of program directors believe that additional training in ophthalmology and other subspecialties should be incorporated into the training programs of primary care physicians. Ophthalmologists and other subspecialists should develop programs at the medical student level directed toward the education of primary care physicians, and should participate in the training of primary care physicians, as well as their own subspecialty residents. In the future, the primary care physician must be considered part of the "eye care team." Enhancing the ophthalmologic education of primary care physicians is in the best interest of quality patient care.