A frequent CYP2D6 variant promotes skipping of exon 3 and reduces CYP2D6 protein expression in human liver samples.

CYP2D6 is one of the most polymorphic drug-metabolizing enzymes in the liver. While genetic CYP2D6 variants serve as clinical biomarkers to predict CYP2D6 activity, large inter-person variability in CYP2D6 expression remains unaccounted for. Previous results suggest that there is variable expression of a CYP2D6 splice isoform with an in-frame deletion of exon 3 (CYP2D6ΔE3) encoding a protein lacking numerous active site residues. Here, using fragment analysis and RT-qPCR, we revealed that rs1058164 G (MAF = 27%-43%) is associated with increased formation of CYP2D6∆E3 in human liver samples (1.4-2.5-fold) and transfected cells. Furthermore, western blots showed that rs1058164 G was associated with a 50% decrease in full-length hepatic CYP2D6 protein expression. In addition, by studying a larger liver cohort, we confirmed our previous results that rs16947 (CYP2D6*2) reduces full-length CYP2D6 mRNA by increasing the production of an unstable splice isoform lacking exon 6 (CYP2D6ΔE6) and that the impact of CYP2D6ΔE6 is offset in carriers of the downstream enhancer variant rs5758550. The three frequent SNPs (rs1058164, rs16947, and rs5758550) form various 3-SNP-haplotypes, each with distinct CYP2D6 expression characteristics. Using an expression score (ES) system, we tested the impact of the 3-SNP-haplotype on improving the standard model to predict hepatic CYP2D6 protein expression based on genotype. A model that incorporates the 3-SNP-haplotype provided the best fit for CYP2D6 expression and also accounted for more variability in CYP2D6 protein levels (59%) than a model based on the accepted standard (36%) or one that only adds rs16947 and rs5758550 (42%). Clinical studies are needed to determine whether including the 3-SNP-haplotype alongside current standard CYP2D6 models improves the predictive value of CYP2D6 panels.

Conditioned social preference and reward value of activating oxytocin-receptor-expressing ventral tegmental area neurons following repeated daily binge ethanol intake.

BACKGROUND: Individuals with alcohol use disorder (AUD) exhibit a disruption of social behavior and dysregulation of oxytocin signaling in the brain, possibly reflecting decreased activation of oxytocin receptors (OxTRs) in reward pathways in response to social stimuli. We hypothesize that daily binge ethanol intake causes a deficit in social reward and oxytocin signaling in the ventral tegmental area (VTA). METHODS: After 9 weeks of daily binge ethanol intake (blood ethanol concentration >80 mg%), OxTR-cre mice underwent conditioned place preference for social reward. Separate groups of mice were tested for the effects of binge ethanol on voluntary social interactions, food reward, locomotion, and anxiety-like behaviors. A subset of mice underwent transfection of OxTR-expressing VTA neurons (VTAOxtr ) with a light-sensitive opsin, followed by operant training to respond to light delivered to VTA. RESULTS: Ethanol-naïve male mice increased the time spent on the side previously paired with novel mice while ethanol-treated mice did not. Binge ethanol did not affect conditioned place preference for food reward in males, but this response was weakened in ethanol-treated females. Ethanol treatment also caused a sex-specific impairment of voluntary social interactions with novel mice. There were minimal differences between groups in measures of anxiety and locomotion. Ethanol-naïve mice had significantly greater operant responding for activation of VTAOxtr than sham-transfected mice but ethanol-treated mice did not. There was no difference in the number of VTAOxtr after binge ethanol. CONCLUSIONS: Daily binge ethanol causes social reward deficits that cannot be explained by nonspecific effects on other behaviors, at least in males. Only ethanol-naïve mice exhibited positive reinforcement caused by activation of VTAOxtr while daily binge ethanol did not alter the number of VTAOxtr in either males or females. Thus, subtle dysregulation of VTAOxtr function may be related to the social reward deficits caused by daily binge ethanol.

Ferric citrate is a safe and digestible source of iron in broilers and piglets.

BACKGROUND: Iron (Fe) is traditionally supplemented in poultry and swine diets using inorganic forms (e.g. sulfates, oxides). However, research suggests that organic sources are more beneficial due to greater bioavailability. In this paper, we present results from four studies aimed at assessing ferric citrate (CI-FER™, Akeso Biomedical Inc., Burlington, MA, USA) as a safe and effective source of Fe for broilers and piglets. METHODS: A total of four studies were performed in Germany following standard farming practices for each species. One study in day-old broiler chicks and one study in weaned piglets were designed as target animal safety studies where animals were randomly allocated to one of three treatment groups: a negative control group, the proposed dose group and a multifold dose group. Broilers and pigs were fed the experimental diets for 35 and 42 days, respectively. In each study, average daily feed intake, average daily weight gain and feed conversion ratio were measured, and blood samples were taken at study end for routine biochemistry and haematology. The other two studies were designed to evaluate different sources of dietary Fe for weaned piglets bred and managed under standard farm conditions. All piglets received routine Fe injections (200 mg Fe dextran, intramuscular) on day 3 of age, as well as the experimental diets for 42 days. In both studies, performance parameters were measured. In one study, Fe digestibility and serum Fe, superoxide dismutase and haptoglobin were also measured. In all studies, the general health status of the animals was monitored daily and all culls and mortality recorded. Each study followed a complete randomised block design. RESULTS: In broilers, ferric citrate was well tolerated up to 2,000 mg/kg feed (×10 the recommended inclusion rate) and no adverse effects on growth, blood parameters or mortality were observed. In piglets, ferric citrate was well tolerated up to 5,000 mg/kg feed (×10 the recommended inclusion rate) with no adverse effects on growth, blood parameters or mortality. In addition, piglets fed ferric citrate performed significantly better than animals fed the negative control diet (containing only endogenous Fe) and those fed inorganic forms of Fe. Moreover, piglets fed ferric citrate demonstrated improved Fe digestibility and improved oxidative status. Altogether, these findings show that ferric citrate is a safe and easily digestible source of dietary Fe for broilers and piglets.

Anthelmintic efficacy on UK Thoroughbred stud farms.

Anthelmintic drugs have been applied indiscriminately to control horse nematodes for over 40 years. We undertook a comprehensive study to investigate efficacy of the four available broad-spectrum anthelmintic drugs on 16 Thoroughbred stud farms using the faecal egg count reduction test. Efficacy against strongyles was determined by calculating the percentage of reduction in faecal egg count between the group mean at Day 0 and Days 14-17 post-treatment and the 95% lower confidence intervals estimated by non-parametric bootstrapping. Individual strongyle faecal egg count reduction tests (n=429) were performed in which 179, 131, 89 and 30 horses were administered ivermectin, moxidectin, pyrantel and fenbendazole, respectively. Moxidectin was efficacious in all tests (faecal egg count reduction range: 99.8-100%; 95% lower confidence intervals range: 96.8-100%) and reduced efficacy of ivermectin (faecal egg count reduction range: 85.7-100%; 95% lower confidence intervals range: 65-100%) was observed in one group of yearlings. Reduced pyrantel efficacy was observed in five groups of yearlings (faecal egg count reduction range: 0-73%; 95% lower confidence intervals range: 0-59.5%), but pyrantel was found to be efficacious when administered to mares (faecal egg count reduction range: 98-99.4%; 95% lower confidence intervals range: 91.8-99.3%). Low efficacy of fenbendazole was always observed (faecal egg count reduction range: 0.4-41%; 95% lower confidence intervals not calculable). Two further methods for estimating efficacy were applied and outputs obtained using all methodologies were in agreement. Efficacy against Parascaris equorum was assessed on four farms: fenbendazole had acceptable efficacy (faecal egg count reduction range: 97.5-99.9%; 95% lower confidence intervals range: 96.3-99.1%), but reduced efficacy of ivermectin was observed (faecal egg count reduction range: 25.5-91.2%; 95% lower confidence intervals range: 6.7-82.4%). Strongyle faecal egg count were analysed at approximately 2 week intervals for up to 12 weeks after anthelmintic drug administration to determine the egg reappearance period for moxidectin, ivermectin and pyrantel. The egg reappearance period for all three anthelmintic drugs was shorter than previously observed. Overall, our results indicate that ivermectin and moxidectin administration provided acceptable efficacy at 14 days; however, egg reappearance period results suggest that these products are working less effectively than measured previously. As shortened egg reappearance period is believed to be an early indicator of resistance, this highlights the issue of impending multi-drug resistance in strongyles on stud farms.