A Sub-Acute Dosing Study of Saxitoxin and Tetrodotoxin Mixtures in Mice Suggests That the Current Paralytic Shellfish Toxin Regulatory Limit Is Fit for Purpose.

Paralytic shellfish poisoning is a worldwide problem induced by shellfish contaminated with paralytic shellfish toxins. To protect human health, a regulatory limit for these toxins in shellfish flesh has been adopted by many countries. In a recent study, mice were dosed with saxitoxin and tetrodotoxin mixtures daily for 28 days showing toxicity at low concentrations, which appeared to be at odds with other work. To further investigate this reported toxicity, we dosed groups of mice with saxitoxin and tetrodotoxin mixtures daily for 21 days. In contrast to the previous study, no effects on mouse bodyweight, food consumption, heart rate, blood pressure, grip strength, blood chemistry or hematology were observed. Furthermore, no histological findings were associated with dosing in this trial. The dose rates in this study were 2.6, 3.8 and 4.9 times greater, respectively, than the highest dose of the previous study. As rapid mortality in three out of five mice was observed in the previous study, the deaths are likely to be due to the methodology used rather than the shellfish toxins. To convert animal data to that used in a human risk assessment, a 100-fold safety factor is required. After applying this safety factor, the dose rates used in the current study were 3.5, 5.0 and 6.5 times greater, respectively, than the acute reference dose for each toxin type set by the European Union. Furthermore, it has previously been proposed that tetrodotoxin be included in the paralytic shellfish poisoning suite of toxins. If this were done, the highest dose rate used in this study would be 13 times the acute reference dose. This study suggests that the previous 28-day trial was flawed and that the current paralytic shellfish toxin regulatory limit is fit for purpose. An additional study, feeding mice a diet laced with the test compounds at higher concentrations than those of the current experiment, would be required to comment on whether the current paralytic shellfish toxin regulatory limit should be modified.

The Effect of Experimental Protocol on the Toxicity of Saxitoxin in Mice.

Regulatory limits for toxins in shellfish are required to ensure the health of consumers. However, these limits also impact the profitability of shellfish industries making it critical that they are fit for purpose. Since human toxicity data is rarely available, the setting of regulatory limits is dependent on animal data which can then be extrapolated for use in the assessment of human risk. The dependence on animal data to keep humans safe means that it is critical that the toxicity data used is robust and of high quality. Worldwide, the protocols used in toxicity testing are varied, making it hard to compare results and adding confusion over which results better reflect the true toxicity. In this study, we look at the effect of mouse gender, i.p. dose volume, mouse body weight and feeding protocols (both acute and sub-acute) on the toxicity of saxitoxin. This allowed the effect of different variables used in toxicity testing to be understood and showed that the feeding protocol used in both acute and sub-acute studies greatly influenced the toxicity of saxitoxin in mice. Therefore, the adoption of a standard protocol for the testing of shellfish toxins is recommended.

Sub-acute feeding study of a tall fescue endophyte in a perennial ryegrass host using mice.

Epichloë endophytes in grass associations express a myriad of secondary metabolites which can affect the health of grazing animals and reduce the impact of insect pests on pasture. The ideal endophyte-grass association must have a favourable chemical profile such that the impact on animal health is minimised while the beneficial, deterrent effect on insect pests is maximised. A number of endophyte-perennial ryegrass associations have been successfully commercialised but research is on-going to further improve production in farming systems. Secondary metabolites expressed by endophyte-infected tall fescue include lolines, an animal-safe class of compound which imparts a potent effect on insects. Since endophyte-infected perennial ryegrass does not express lolines, a tall fescue endophyte, AR501, was inoculated into perennial ryegrass in an attempt to improve the insect resistance of this pasture type. In addition to animal safety, it is imperative that consideration is given to the safety of humans consuming animal products derived from livestock grazing the novel pasture. Although pure loline alkaloids have previously been tested on mice it is essential that the entire AR501 endophyte-infected perennial ryegrass matrix is tested since this will result in the exposure of both known and unknown secondary metabolites to mice. Three treatment groups each containing 6 male and 6 female mice were fed diets containing AR501 endophyte-infected perennial ryegrass seed (30%), perennial ryegrass seed containing no endophyte (30%) or a diet without seed (control) for 3 weeks. Mice fed control diet ate more than either of the treatment groups fed a diet containing seed. Male mice fed diet containing Nil endophyte seed ate more than those eating AR501 endophyte-infected perennial ryegrass seed although there was no difference observed in the food intake of female mice. While a few statistically significant differences were observed in the haematology and serum biochemical data, in every instance the difference was restricted to only one gender so is considered unlikely to be of toxicological significance. Mice fed AR501 endophyte-infected perennial ryegrass seed remained healthy throughout the experimental period despite consuming 62,000 mg/kg lolines and 4600 mg/kg peramine per day as well as the wide array of other unknown secondary metabolites expressed by this endophyte. Although animal products may contain additional metabolites as a result of animal metabolism, this experiment raises no food safety concerns for AR501 endophyte-infected perennial ryegrass.

Sub-Acute Feeding Study of Saxitoxin to Mice Confirms the Effectiveness of Current Regulatory Limits for Paralytic Shellfish Toxins.

Regulatory limits for shellfish toxins are required to protect human health. Often these limits are set using only acute toxicity data, which is significant, as in some communities, shellfish makes up a large proportion of their daily diet and can be contaminated with paralytic shellfish toxins (PSTs) for several months. In the current study, feeding protocols were developed to mimic human feeding behaviour and diets containing three dose rates of saxitoxin dihydrochloride (STX.2HCl) were fed to mice for 21 days. This yielded STX.2HCl dose rates of up to 730 µg/kg bw/day with no effects on food consumption, growth, blood pressure, heart rate, motor coordination, grip strength, blood chemistry, haematology, organ weights or tissue histology. Using the 100-fold safety factor to extrapolate from animals to humans yields a dose rate of 7.3 µg/kg bw/day, which is well above the current acute reference dose (ARfD) of 0.5 µg STX.2HCl eq/kg bw proposed by the European Food Safety Authority. Furthermore, to reach the dose rate of 7.3 µg/kg bw, a 60 or 70 kg human would have to consume 540 or 630 g of shellfish contaminated with PSTs at the current regulatory limit (800 µg/kg shellfish flesh), respectively. The current regulatory limit for PSTs therefore seems appropriate.

Development of social contact and loneliness measures with validation in social prescribing.

AIMS: This paper describes two patient-reported measures of social contact and loneliness, which are closely related concepts. The first measure (R-Outcomes Social Contact measure) was developed from scratch, based on customer needs and literature review. It covers emotional and social aspects using positive terms. The second measure (R-Outcomes Loneliness measure) is adapted from the GSS Loneliness Harmonised Standard. Both measures are patient-reported outcome measures, based on patients' own perception of how they feel. METHOD: This development started in 2016 in response to customers' requests to measure social contact/loneliness for patients in social prescribing projects.Both measures are compared with three other loneliness measures (the GSS Loneliness Harmonised Standard, De Jong Gierveld and Campaign to End Loneliness). Both measures are short (36 and 21 words, respectively). Mean improvement is reported as a positive number on a 0-100 scale (where high is good).We tested the psychometric performance and construct validity of the R-Outcomes Social Contact measure using secondary analysis of anonymised data collected before and after social prescribing interventions in one part of Southern England. RESULTS: In the validation study, 728 responses, collected during 2019-2020, were analysed. 90% were over 70 years old and 62% women. Cronbach's α=0.76, which suggests that it is appropriate to use a single summary score. Mean Social Contact scores before and after social prescribing intervention were 59.9 (before) and 66.7 (after, p<0.001).Exploratory factor analysis shows that measures for social contact, health status, health confidence, patient experience, personal well-being, medication adherence and social determinants of health are correlated but distinct factors. Construct validation shows that the results are consistent with nine hypotheses, based on the loneliness literature. CONCLUSION: The R-Outcomes Social Contact measure has good psychometric and construct validation results in a population referred to social prescribing. It is complementary to other R-Outcomes measures.

Identification and Structure Elucidation of Epoxyjanthitrems from Lolium perenne Infected with the Endophytic Fungus Epichloë festucae var. lolii and Determination of the Tremorgenic and Anti-Insect Activity of Epoxyjanthitrem I.

Epoxyjanthitrems I-IV (1-4) and epoxyjanthitriol (5) were isolated from seed of perennial ryegrass (Lolium perenne) infected with the endophytic fungus Epichloë festucae var. lolii. Although structures for epoxyjanthitrems I-IV have previously been proposed in the literature, this is the first report of a full structural elucidation yielding NMR (Nuclear magnetic resonance) assignments for all five epoxyjanthitrem compounds, and additionally, it is the first isolation of epoxyjanthitriol (5). Epoxyjanthitrem I induced tremors in mice and gave a dose dependent reduction in weight gain and feeding for porina (Wiseana cervinata), a common pasture pest in New Zealand. These data suggest that epoxyjanthitrems are involved in the observed effects of the AR37 endophyte on livestock and insect pests.

Rhodium(III)-catalyzed C-H activation/annulation with vinyl esters as an acetylene equivalent.

The behavior of electron-rich alkenes in rhodium-catalyzed C-H activation/annulation reactions is investigated. Vinyl acetate emerges as a convenient acetylene equivalent, facilitating the synthesis of sixteen 3,4-unsubstituted isoquinolones, as well as select heteroaryl-fused pyridones. The complementary regiochemical preferences of enol ethers versus enol esters/enamides is discussed.

Stereoselective glycosylations using oxathiane spiroketal glycosyl donors.

Novel oxathiane spiroketal donors have been synthesised and activated via an umpolung S-arylation strategy using 1,3,5-trimethoxybenzene and 1,3-dimethoxybenzene. The comparative reactivity of the resulting 2,4,6-trimethoxyphenyl (TMP)- and 2,4-dimethoxyphenyl (DMP)-oxathiane spiroketal sulfonium ions is discussed, and their α-stereoselectivity in glycosylation reactions is compared to the analogous TMP- and DMP-sulfonium ions derived from an oxathiane glycosyl donor bearing a methyl ketal group. The results show that the stereoselectivity of the oxathiane glycosyl donors is dependent on the structure of the ketal group and reactivity can be tuned by varying the substituent on the sulfonium ion.

Discovery of isoindoline and tetrahydroisoquinoline derivatives as potent, selective PPARδ agonists.

Small molecule isoindoline and tetrahydroisoquinoline derivatives have been identified as selective agonists of human peroxisome proliferator-activated receptor δ (PPARδ. Compound 18 demonstrated efficacy in a biomarker for increased fatty acid oxidation, with upregulation of pyruvate dehydrogenase kinase, isozyme 4 (PDK4) in human primary myotubes.