Ecological Toxicity Methods and Metals. An examination of two case studies (8 pp + 1)

Goal, Scope and Background The Apeldoorn Workshop (April 15th, 2004, Apeldoorn, NL) brought together specialists in LCA and Risk Assessment to discuss current practices and complications of the life cycle impact assessment (LCIA) ecological toxicity (ecotox) methodologies for metals. The consensus was that the LCIA methods currently available do not appropriately characterize impacts of metals due to lack of fundamental metals chemistry in the models. A review of five methods available to perform ecotox impact assessment for metals has been prepared to provide Life Cycle Assessment (LCA) practitioners with a better understanding of the current state of the science and potential biases related to metals. The intent is to provide awareness on issues related to ecotox impact assessment. Methods In this paper two case studies, one a copper based product (copper tube), the other a zinc-based product (gutter systems), were selected and examined by applying freshwater ecological toxicity impact models – USES-LCA, Eco-indicator 99 (EI 99), IMPACT 2002, EDIP 97, and CalTOX-ETP. Both studies are recent, comprehensive, cradle-to-gate, and peer-reviewed. The objective is to review the LCIA results in the context of the practical concerns identified by the Apeldoorn Declaration, in particular illustrating any inconsistencies such as chemical characterization coverage, species specificity, and relative contribution to impact results. Results and Discussion The results obtained from all five of the LCIA methods for the copper tube LCI pointed to the same substance as being the most important – copper. This result was obtained despite major fundamental differences between the LCIA methods applied. However, variations of results were found when examining the freshwater ecological toxicity potential of zinc gutter systems. Procedural difficulties and inconsistencies were observed. In part this was due to basic differences in model nomenclature and differences in coverage (IMPACT 2002+ and EDIP 97 contained characterization factors for aluminium that resulted in 90% and 22% contribution to burden respectively, the other three methods did not). Differences were also observed relative to the emissions source compartment. In the case of zinc, air emissions were found to be substantial for some ecotox models, whereas, water emissions results were found to be of issue for others. Conclusions This investigation illustrates the need to proceed with caution when applying LCIA ecotox methodologies to life cycle studies that include metals. Until further improvements are made, the deficiencies should be clearly communicated as part of LCIA reporting. Business or policy decisions should not without further discussion be based solely on the results of the currently available methods for assessing ecotoxicity in LCIA. Outlook The outlook to remedy deficiencies in the ecological toxicity methods is promising. Recently, the LCIA Toxic Impacts Task Force of the UNEP/SETAC Life Cycle Initiative has formed a subgroup to address specific issues and guide the work towards establishment of sound characterization factors for metals. Although some measure of precision of estimation of potential impact has been observed, such as in the case of copper, accuracy is also a major concern and should be addressed. Further investigation through controlled experimentation is needed, particularly LCIs composed of a variety of inorganics as well as organics constituents. Support for this activity has come from the scientific community and industry as well. Broader aspects of structure and nomenclature are being collectively addressed by the UNEP/SETAC Life Cycle Initiative. These efforts will bring practical solutions to issues of naming conventions and LCI to LCIA flow assignments.     

The Fes/Fer non-receptor tyrosine kinase cooperates with Src42A to regulate dorsal closure in Drosophila

Fes/Fer non-receptor tyrosine kinases regulate cell adhesion and cytoskeletal reorganisation through the modification of adherens junctions. Unregulated Fes/Fer kinase activity has been shown to lead to tumours in vivo. Here, we show that Drosophila Fer localises to adherens junctions in the dorsal epidermis and regulates a major morphological event, dorsal closure. Mutations in Src42A cause defects in dorsal closure similar to those seen in dfer mutant embryos. Furthermore, Src42A mutations enhance the dfer mutant phenotype, suggesting that Src42A and DFer act in the same cellular process. We show that DFer is required for the formation of the actin cable in leading edge cells and for normal rates of dorsal closure. We have isolated a gain-of-function mutation in dfer (dfergof) that expresses an N-terminally fused form of the protein, similar to oncogenic forms of vertebrate Fer. dfergof blocks dorsal closure and causes axon misrouting. We find that in dfer loss-of-function mutants beta-catenin is hypophosphorylated, whereas in dfergof beta-catenin is hyperphosphorylated. Phosphorylated beta-catenin is removed from adherens junctions and degraded, thus implicating DFer in the regulation of adherens junctions.     

Preferential production of prostaglandin D2 by lipopolysaccharide stimulated human choriodecidual explants

It has been postulated that the progression of a pregnancy to term is, in part, the result of a relative maternal Th2 immunological state. Prostaglandins (PG) are critical mediators throughout pregnancy. Recent studies have demonstrated that one PG, PGD2, may be a mediator of a Th2 immunological state. To date, very little is known about the factors that regulate of PGD2 production by human gestational tissues. Placentae were collected from women undergoing Caesarean sections at term. Amnion was separated from the choriodecidua and choriodecidual explants established. Explants were allowed to equilibrate overnight in media containing 10% fetal calf serum. The following day, media were replaced with serum free media and then after an additional 24-h, media were collected and the wet weight of the tissues determined. Production rates of PGs were determined using radioimmunoassays. At all concentrations tested, LPS significantly enhanced PGD2 production by human choriodecidual explants compared to PGE2 and PGF2alpha production. Neutralization of TNF-alpha and IL-10 further increased the production of LPS stimulated PGD2 production. We suggest that a novel stimulatory pathway that drives the production of PGD2 has been uncovered.     

Differential roles of the co-stimulatory molecules GITR and CTLA-4 in the immune response to Trichinella spiralis

We investigated the roles of the regulatory molecules glucocorticoid-induced TNF receptor family-related protein (GITR) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) in murine infection with the nematode parasite Trichinella spiralis. Expression of GITR and CTLA-4 was rapidly upregulated on cells in the mesenteric lymph nodes and spleen, with approximately 80% of CD4+ lymphocytes expressing GITR by day 7 post-infection, coinciding with release and dissemination of newborn larvae. As the infection progressed to the chronic muscle phase, expression of GITR returned to normal, whereas CTLA-4 was sustained as late as day 60. Mice treated with anti-GITR antibodies rapidly developed higher titres of parasite-specific IgG1, IgG2a, IgG2b and IgM than controls. This was accompanied by elevated background lymphocyte proliferation, but parasite establishment in the intestine or the muscle was unaffected. In contrast, treatment with anti-CTLA-4 antibody resulted in elevated serum IgE, enhanced production of interleukin-4 and interleukin-10, and lower numbers of parasites recovered from skeletal muscle. These results reveal different temporal and regulatory roles for CTLA-4 and GITR in immune responses to helminth infection.     

Cone contrast computations: physical versus perceived background and colour constancy

The light reflected from an object depends both on the object's surface and on the illuminant. Visual systems attempt to resolve this intrinsic ambiguity by comparing the light reflected from the object with respect to the background by computing the difference between the object-background light sampled by three cones. The cone-contrasts for the sample-background stimulus under the test illumination should correspond to the cone-contrasts for samples matched in appearance under the standard background (C). The validity of this cone-contrast rule (Whittle, 2003) and its possible link with stability of perceived colour was studied here using six test illuminants. A successive asymmetric colour-matching task with 40 simulated Munsell samples (value 7, chroma 4) on a neutral background (N7) was used. The subject adjusted the sample under standard illuminant C to match the colour appearance of the test sample under one of the test illuminants. Brunswik Ratio (BR) was used as an index of stability of colour appearance. When computed with respect to the reference illuminant C, the cone contrast rule was violated (particularly for S-cone-contrast). However, if a new reference point based on the perceived colour of the neutral background under the test illumination was used, the cone contrast rule applied. That is, when cone contrasts of the matching samples are computed with respect to this perceived background, they correspond to cone contrasts of the test stimuli. This represents a form of discounting the illuminant for the purpose of determining an object's cone-contrast against the background, which does not vary with background illumination. These cone contrasts, however, do not provide any information about the colour appearance of objects under particular viewing conditions, unless calibrated against a standard by allowing subjects to learn particular colours.     

A role for cyclic nucleotide monophosphates in synaptic modulation by a crayfish neuropeptide

DF2 (DRNFLRFamide), a FMRFamide-like peptide, has been shown to increase the amount of transmitter released at crayfish neuromuscular junctions. Here, we examined a possible role for the cyclic nucleotide monophosphates, cAMP and cGMP, in DF2's effects on synaptic transmission. The effects of DF2 on synaptic transmission were monitored by recording excitatory postsynaptic potentials (EPSPs) in the deep abdominal extensor muscles of the crayfish, Procambarus clarkii. A number of activators and inhibitors were used to determine whether or not cAMP, cGMP, protein kinase A (PKA) and protein kinase G (PKG) mediate the effect of this neuropeptide. Phosphodiesterase inhibitors, known to inhibit the breakdown of cAMP (IBMX) and/or cGMP (mdBAMQ), potentiate the effect of DF2 on synaptic transmission. Activators of PKA (Sp-cAMPS) and PKG (8-pCPT-cGMP) increase EPSP amplitude, mimicking the effects of DF2. Inhibitors of PKA (Rp-cAMPS) and PKG (Rp-8-pCPT-cGMPS) each block a portion of the response to the peptide, and when applied together these two inhibitors completely block the response. Taken together, these results indicate that cyclic nucleotides and cyclic nucleotide-dependent protein kinases are necessary components of the pathway underlying modulation by this neuropeptide.     

Modelling environmental variation in Young's modulus for Pinus radiata and implications for determination of critical buckling height

• Background and Aims Although density-specific stiffness,E/, (where E is Young's modulus and is wood density) is oftenassumed constant by the elastic similarity model, and in determinationof critical buckling height (H_crit), few studies have testedthis assumption within species. Here this assumption is testedfor Pinus radiata growing across an environmental gradient,and theory is combined with data to develop a model of Young'smodulus. • Methods Analyses use an extensive series of environmentalplots covering the range of climatic and edaphic conditionsover which P. radiata is grown in New Zealand. Reduced majoraxis regression was used to determine scaling exponents betweenlog–log plots of H_crit vs. groundline diameter (D), andE/ vs. D. Path analysis was used to identify significant directand indirect (through stem slenderness) edaphic and climaticinfluences on E. • Key Results Density-specific stiffness exhibited 3-foldvariation. As E/ scaled positively with D, the exponent of 0·95between H_crit and D exceeded the assumed value of 0·67under constant E/. The final path analysis model included meanair temperature in early autumn (T_aut) and slenderness as significant(P < 0·05) positive direct influences on E. Tree leafarea index and T_aut were indirectly associated with E throughtheir significant (P < 0·05) positive direct relationshipwith stem slenderness. Young's modulus was most sensitive toT_aut, followed by stem slenderness then leaf area index, andthe final model explained 76 % of the variance in E. • Conclusions The findings suggest that within speciesE/ variation may influence H_crit and the scaling exponent betweenD and H_crit so important in assumptions regarding allometricrelationships. The model presented may provide a useful meansof determining variation in E, E/ and H_crit across environmentalgradients.     

Thrombin activates AMP-activated protein kinase in endothelial cells via a pathway involving Ca2+/calmodulin-dependent protein kinase kinase beta

AMP-activated protein kinase (AMPK) is a sensor of cellular energy state in response to metabolic stress and other regulatory signals. AMPK is controlled by upstream kinases which have recently been identified as LKB1 or Ca2+/calmodulin-dependent protein kinase kinase beta (CaMKKbeta). Our study of human endothelial cells shows that AMPK is activated by thrombin through a Ca2+-dependent mechanism involving the thrombin receptor protease-activated receptor 1 and Gq-protein-mediated phospholipase C activation. Inhibition of CaMKK with STO-609 or downregulation of CaMKKbeta using RNA interference decreased thrombin-induced AMPK activation significantly, indicating that CaMKKbeta was the responsible AMPK kinase. In contrast, downregulation of LKB1 did not affect thrombin-induced AMPK activation but abolished phosphorylation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleoside. Thrombin stimulation led to phosphorylation of acetyl coenzyme A carboxylase (ACC) and endothelial nitric oxide synthase (eNOS), two downstream targets of AMPK. Inhibition or downregulation of CaMKKbeta or AMPK abolished phosphorylation of ACC in response to thrombin but had no effect on eNOS phosphorylation, indicating that thrombin-stimulated phosphorylation of eNOS is not mediated by AMPK. Our results underline the role of Ca2+ as a regulator of AMPK activation in response to a physiologic stimulation. We also demonstrate that endothelial cells possess two pathways to activate AMPK, one Ca2+/CaMKKbeta dependent and one AMP/LKB1 dependent.     

Indigenous Knowledge of Rock Kangaroo Ecology in Western Arnhem Land, Australia

Indigenous peoples of western Arnhem Land, central northern Australia, have detailed knowledge of the rock kangaroos of the region, species that are little known to science. Information about the ecology of the species is required for their conservation and management. Ethnoecological studies can assist senior indigenous people with transfer of knowledge and can give respect and meaningful employment to those involved. We used semidirected interviews in the regional vernacular, Bininj Kunwok, to record indigenous knowledge of the ecology of the four rock kangaroo species (Petrogale brachyotis, P. concinna, Macropus bernardus and M. robustus). Discussions focussed on habitat preferences, diet, activity patterns, reproduction, predation, and hunting practices. The ethnoecological knowledge of the rock kangaroo species was extensive, and both complemented and extended that reported in the scientific literature. In contrast to scientific understanding of taxonomy and ecology, consultants recognized the rock kangaroos as a natural group. They also described subtle differences in the species’ comparative ecology. The methodology used proved highly successful and we recommend recording indigenous knowledge of the ecology of fauna species in the local vernacular wherever possible. This study is one of the most comprehensive ethnozoological studies of a group of species undertaken in Australia.