Dying for change: A roadmap to refine the fish acute toxicity test after 40 years of applying a lethal endpoint

The fish acute toxicity test (TG203; OECD, 2019) is frequently used and highly embedded in hazard and risk assessment globally. The test estimates the concentration of a chemical that kills 50% of the fish (LC50) over a 96 h exposure and is considered one of the most severe scientific procedures undertaken. Over the years, discussions at the Organisation for Economic Co-operation and Development (OECD) have resulted in changes to the test which reduce the number of fish used, as well as the development of a (potential) replacement test (TG236, OECD, 2013). However, refinement of the mortality endpoint with an earlier (moribundity) endpoint was not considered feasible during the Test Guideline’s (TG) last update in 2019. Several stakeholders met at a UK-based workshop to discuss how TG203 can be refined, and identified two key opportunities to reduce fish suffering: (1) application of clinical signs that predict mortality and (2) shortening the test duration. However, several aspects need to be addressed before these refinements can be adopted. TG203 has required recording of major categories of sublethal clinical signs since its conception, with the option to record more detailed signs introduced in the 2019 update. However, in the absence of guidance, differences in identification, recording and reporting of clinical signs between technicians and laboratories is likely to have generated piecemeal data of varying quality. Harmonisation of reporting templates, and training in clinical sign recognition and recording are needed to standardise clinical sign data. This is critical to enable robust data-driven detection of clinical signs that predict mortality. Discussions suggested that the 96 h duration of TG203 cannot stand up to scientific scrutiny. Feedback and data from UK contract research organisations (CROs) conducting the test were that a substantial proportion of mortalities occur in the first 24 h. Refinement of TG203 by shortening the test duration would reduce suffering (and test failure rate) but requires a mechanism to correct new results to previous 96 h LC50 data. The actions needed to implement both refinement opportunities are summarised here within a roadmap. A shift in regulatory assessment, where the 96 h LC50 is a familiar base for decisions, will also be critical

The Hans Tausen drill: design, performance, further developments and some lessons learned

International audienceIn the mid-1990s, excellent results from the GRIP and GISP2 deep drilling projects in Greenland opened up funding for continued ice-coring efforts in Antarctica (EPICA) and Greenland (NorthGRIP). The Glaciology Group of the Niels Bohr Institute, University of Copenhagen, was assigned the task of providing drilling capability for these projects, as it had done for the GRIP project. The group decided to further simplify existing deep drill designs for better reliability and ease of handling. The drill design decided upon was successfully tested on Hans Tausen Ice Cap, Peary Land, Greenland, in 1995. The 5.0 m long Hans Tausen (HT) drill was a prototype for the ∼11 m long EPICA and NorthGRIP versions of the drill which were mechanically identical to the HT drill except for a much longer core barrel and chips chamber. These drills could deliver up to 4 m long ice cores after some design improvements had been introduced. The Berkner Island (Antarctica) drill is also an extended HT drill capable of drilling 2 m long cores. The success of the mechanical design of the HT drill is manifested by over 12 km of good-quality ice cores drilled by the HT drill and its derivatives since 1995

Specific stimulation of migration of human keratinocytes by mu-opiate receptor agonists

There are several indications that neuropeptides, especially the opiate receptor agonists, modulate the immune response by stimulating the formation of granulation tissue and enhancing the reepithelialization. We observed that the mu-opiate receptor ligand beta-endorphin stimulates the migration of cultured human foreskin keratinocytes. After 1 hour exposure to 1 microM beta-endorphin, the keratinocytes experienced an increase of cell diameter by cellular elongation and stimulation of migration. Dynorphin had a lesser effect under the same condition. The opiate receptor antagonist naltrexone significantly reduced the effect of beta-endorphin on keratinocyte migration. This migratory effect of mu-opiate receptor agonists in vitro indicates that the opioid peptides, released in wounds, could play a key role in the final reepithelialization and tissue regeneration in wound healing. This new knowledge will help us not only to understand the mechanism of wound healing but also to improve the therapeutic strategy in the healing of painful chronic wounds

Expression of mu-opiate receptor in human epidermis and keratinocytes

There is increasing evidence that neurotransmitters play a crucial role in skin physiology and pathology. The expression and production of proopiomelanocortin molecules such as beta-endorphin in human epidermis suggest that an opiate receptor is present in keratinocytes. In this paper we show that human epidermal keratinocytes express a mu-opiate receptor on both the mRNA level and the protein level. Performing polymerase chain reaction with cDNA libraries from human epidermal keratinocytes gave the polymerase chain reaction products of the expected length, which were confirmed as mu-opiate receptors by Southern blot analysis. Using in situ hybridization techniques with a specific probe for mu-opiate receptors we detected the receptor in human epidermis. There was a cytoplasmic expression in all layers of the epidermis, which was more distinct in the suprabasal layers. Immunohistochemistry using the mu-opiate receptor-specific antibody indicates that epidermis expresses protein as well, and that the protein level is more elevated in the basal layer. The correlation between the locations of both mRNA and protein expression in skin indicates that the mu-opiate receptor has not only been transcribed but also has a specific function. To prove a function of the receptor we performed a functional assay using skin organ cultures from human skin transplants. After 48 h incubation with Naloxone or beta-endorphin the expression of the mu-opiate receptor in epidermis was significantly downregulated compared with the control. These results show that a functional receptor indeed exists in human epidermis

Expression of mu-opiate receptor in human epidermis and keratinocytes

There is increasing evidence that neurotransmitters play a crucial role in skin physiology and pathology. The expression and production of proopiomelanocortin molecules such as beta-endorphin in human epidermis suggest that an opiate receptor is present in keratinocytes. In this paper we show that human epidermal keratinocytes express a mu-opiate receptor on both the mRNA level and the protein level. Performing polymerase chain reaction with cDNA libraries from human epidermal keratinocytes gave the polymerase chain reaction products of the expected length, which were confirmed as mu-opiate receptors by Southern blot analysis. Using in situ hybridization techniques with a specific probe for mu-opiate receptors we detected the receptor in human epidermis. There was a cytoplasmic expression in all layers of the epidermis, which was more distinct in the suprabasal layers. Immunohistochemistry using the mu-opiate receptor-specific antibody indicates that epidermis expresses protein as well, and that the protein level is more elevated in the basal layer. The correlation between the locations of both mRNA and protein expression in skin indicates that the mu-opiate receptor has not only been transcribed but also has a specific function. To prove a function of the receptor we performed a functional assay using skin organ cultures from human skin transplants. After 48 h incubation with Naloxone or beta-endorphin the expression of the mu-opiate receptor in epidermis was significantly downregulated compared with the control. These results show that a functional receptor indeed exists in human epidermis

Different expression of mu-opiate receptor in chronic and acute wounds and the effect of beta-endorphin on transforming growth factor beta type II receptor and cytokeratin 16 expression

There is evidence that neuropeptides, especially the opiate receptor agonists, are involved in wound healing. We have previously observed that beta-endorphin, the endogenous ligand for the mu-opiate receptor, stimulates the expression of cytokeratin 16 in a dose-dependent manner in human skin organ cultures. Cytokeratin 16 is expressed in hyperproliferative epidermis such as psoriasis and wound healing. Therefore we were interested to study whether epidermal mu-opiate receptor expression is changed at the wound margins in acute and chronic wounds. Using classical and confocal microscopy, we were able to compare the expression level of mu-opiate receptors and the influence of beta-endorphin on transforming growth factor beta type II receptor in organ culture. Our results show indeed a significantly decreased expression of mu-opiate receptors on keratinocytes close to the wound margin of chronic wounds compared to acute wounds. Additionally beta-endorphin upregulates the expression of transforming growth factor beta type II receptor in human skin organ cultures. These results suggest a crucial role of opioid peptides not only in pain control but also in wound healing. Opioid peptides have already been used in animal models in treatment of wounds; they induce fibroblast proliferation and growth of capillaries, and accelerate the maturation of granulation tissue and the epithelization of the defect. Furthermore opioid peptides may fine-tune pain and the inflammatory response while healing takes place. This new knowledge could potentially be used to design new locally applied drugs to improve the healing of painful chronic wounds