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In drug metabolism studies in horses, non-targeted analysis by means of liquid chromatography coupled with high-resolution mass spectrometry with data-dependent acquisition (DDA) has recently become increasingly popular for rapid identification of potential biomarkers in post-administration biological samples. However, the most commonly encountered problem is the presence of highly abundant interfering components that co-elute with the target substances, especially if the concentrations of these substances are relatively low. In this study, we evaluated the possibility of expanding DDA coverage for the identification of drug metabolites by applying intelligently generated exclusion lists (ELs) consisting of a set of chemical backgrounds and endogenous substances. Daprodustat was used as a model compound because of its relatively lower administration dose (100 mg) compared to other hypoxia-inducible factor stabilizers and the high demand in the detection sensitivity of its metabolites at the anticipated lower concentrations. It was found that the entire DDA process could efficiently identify both major and minor metabolites (flagged beyond the pre-set DDA threshold) in a single run after applying the ELs to exclude 67.7-99.0% of the interfering peaks, resulting in a much higher chance of triggering DDA to cover the analytes of interest. This approach successfully identified 21 metabolites of daprodustat and then established the metabolic pathway. It was concluded that the use of this generic intelligent "DDA + EL" approach for non-targeted analysis is a powerful tool for the discovery of unknown metabolites, even in complex plasma and urine matrices in the context of doping control., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

IOX4, a hypoxia-inducible factor stabilizer, is classified as a banned substance for horses in both horse racing and equestrian sports. We recently reported the pharmacokinetic profiles of IOX4 in horse plasma and urine and also identified potential monitoring targets for the doping control purpose. In this study, a long-term longitudinal analysis of IOX4 in horse hair after a nasoesophageal administration of IOX4 (500 mg/day for 3 days) to three thoroughbred mares is presented for the first time for controlling the abuse/misuse of IOX4. Six bunches of mane hair were collected at 0 (pre), 1, 2, 3, and 6 month(s) postadministration. Our results showed that the presence of IOX4 was identified in all postadministration horse hair samples, but no metabolite could be detected. The detection window for IOX4 could achieve up to 6-month postadministration (last sampling point) by monitoring IOX4 in hair. In order to evaluate the longitudinal distribution of IOX4 over 6 months, a validated quantification method of IOX4 in hair was developed for the analysis of the postadministration samples. Segmental analysis of 2-cm cut hair across the entire length of postadministration hair showed that IOX4 could be quantified up to the level of 1.84 pg/mg. In addition, it was found that the movement of the incorporated IOX4 band in the hair shaft over 6 months varied among the three horses due to individual variation and a significant diffusion of IOX4 band up to 10 cm width was also observed in the 6-month postadministration hair samples., (© 2022 John Wiley & Sons, Ltd.)

Processed pseudogenes, also known as retrocopy genes, are copies of messenger RNAs that have been reverse transcribed into DNA and inserted into the genome. In this study, we identified 62 processed pseudogene candidates as intron-less genes from whole-genome sequencing (WGS) data of Thoroughbred horses using delly structural variation software. The 62 processed pseudogene candidates were confirmed by PCR amplification of intron-less products. A total of 11 processed pseudogenes were confirmed in the genome of all 23 analysed horses, whereas three processed pseudogenes with structures of ATP11B, DPH3 and RPL17 were detected in only one of 115 horses by PCR amplification of intron-less products. Currently, most of the gene doping tests proposed in human and horse sports are adapted PCR-based methods using hydrolysis probes to detect exon/exon junctions in transgenes because the operation is simple and economical. However, when the pseudogene is present in the host genome, the PCR-based methods may have a potential risk of detecting false positives. In this study, because processed pseudogenes that exist less frequently in the horse genome may affect PCR-based transgene detection in gene-doping tests, we propose and demonstrate that PCR amplification and sequencing using primers designed on transgene and promotors and/or polyadenylation signal for gene expression are useful for gene-doping detection as an additional confirmatory test to prevent false positives., (© 2022 Stichting International Foundation for Animal Genetics.)

Gene doping is prohibited for fair competition in human and horse sports. One style of gene doping is the administration of an exogeneous gene, called a transgene, to postnatal humans and horses. Although many transgene detection methods based on quantitative polymerase chain reaction (PCR), including real-time PCR and digital PCR, have been recently developed, it remains difficult to reliably detect low-copy transgenes. In this study, we developed and validated a nested digital PCR method to specifically detect low-copy transgenes. The nested digital PCR consists of (1) preamplification using conventional PCR and (2) droplet digital PCR detection using a hydrolysis probe. Using 5, 10, 20, 60 and 120 transgene copies as template, 496.0, 1089.7, 1820.7, 4313.3 and 7840.0 copies per microlitre, respectively, were detected using our nested digital PCR. Although high concentrations of phenol, proteinase K, ethanol, EDTA, heparin and genomic DNA all inhibited preamplification, their effects on the digital PCR detection were limited. Once preamplification was successful, even substitution of bases within the primers and probes had minimal effects on transgene detection. The nested digital PCR developed in this study successfully detected low-copy transgenes and can be used to perform a qualitative test, indicating its usefulness in the prevention of false positives and false negatives in gene-doping detection., (© 2021 John Wiley & Sons, Ltd.)

Nicotine is classified as a stimulant, and its use is banned in horse racing and equestrian sports by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale, respectively. Because nicotine is a major alkaloid of tobacco leaves, there is a potential risk that doping control samples may be contaminated by tobacco cigarettes or smoke during sample collection. In order to differentiate the genuine doping and sample contamination with tobacco leaves, it is necessary to monitor unique metabolites as biomarkers for nicotine administration and intake. However, little is known about the metabolic fate of nicotine in horses. This is the first report of comprehensive metabolism study of nicotine in horses. Using liquid chromatography/electrospray ionization high-resolution mass spectrometry, we identified a total of 17 metabolites, including one novel horse-specific metabolite (i.e., 4-hydroxy-4-(3-pyridyl)-N-methylbutanamide), in post-administration urine samples after nasoesophageal administration of nicotine to three thoroughbred mares; eight of these compounds were confirmed based on reference standards. Among these metabolites, N-hydroxymethylnorcotinine was the major urinary metabolite in equine, but it could only be tentatively identified by mass spectral interpretation due to the lack of reference material. In addition, we developed simultaneous quantification methods for the eight target analytes in plasma and urine, and applied them to post-administration samples to establish elimination profiles of nicotine and its metabolites. The quantification results revealed that trans-3'-hydroxycotinine could be quantified for the longest period in both plasma (72 h post-administration) and urine (96 h post-administration). Therefore, this metabolite is the most appropriate monitoring target for nicotine exposure for the purpose of doping control due to its long detection times and the availability of its reference material. Further, we identified trans-3'-hydroxycotinine as a unique biomarker allowing differentiation between nicotine administration and sample contamination with tobacco leaves., (Copyright © 2022 Elsevier B.V. All rights reserved.)

IOX4 is a hypoxia-inducible factor prolyl hydroxylase (HIF-PHD) inhibitor, which was developed for the treatment of anemia by exerting hematopoietic effects. The administration of HIF-PHD inhibitors such as IOX4 to horses is strictly prohibited by the International Federation of Horseracing Authorities and the Fédération Équestre Internationale. To the best of our knowledge, this is the first comprehensive metabolic study of IOX4 in horse plasma and urine after a nasoesophageal administration of IOX4 (500 mg/day, 3 days). A total of four metabolites (three mono-hydroxylated IOX4 and one IOX4 glucuronide) were detected from the in vitro study using homogenized horse liver. As for the in vivo study, post-administration plasma and urine samples were comprehensively analyzed with liquid chromatography/electrospray ionization high-resolution mass spectrometry to identify potential metabolites and determine their corresponding detection times. A total of 10 metabolites (including IOX4 glucuronide, IOX4 glucoside, O-desbutyl IOX4, O-desbutyl IOX4 glucuronide, four mono-hydroxylated IOX4, N-oxidized IOX4, and N-oxidized IOX4 glucoside) were found in urine and three metabolites (glucuronide, glucoside, and O-desbutyl) in plasma. Thus, the respective quantification methods for the detection of free and conjugated IOX4 metabolites in urine and plasma with a biphase enzymatic hydrolysis were developed and applied to post-administration samples for the establishment of elimination profiles of IOX4. The detection times of total IOX4 in urine and plasma could be successfully prolonged to at least 312 h., (© 2021 John Wiley & Sons, Ltd.)

In human and equestrian sporting events, one method of gene doping is the illegal use of therapeutic oligonucleotides to alter gene expression. In this study, we aimed to identify therapeutic oligonucleotides via sequencing using matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS). As a model of therapeutic oligonucleotides, 22 bp-long phosphorothioated oligonucleotides (PSOs) were used. By using a Clarity OTX kit for extracting short-length oligonucleotides, a spectrum of singly charged PSO with a mean intensity of 6.08 × 10 4 (standard deviation: 4.34 × 10 3 ) was detected from 500 pmol PSO in 1 ml horse plasma using the linear negative mode of MALDI-TOF MS. In addition, a 17 bp sequence was determined using in-source decay (ISD) mode, indicating that 500 pmol of a PSO in 1 ml plasma is the detection limit for sequencing. Using the determined sequences (17 bp), a targeted gene for PSO was singly identified on the horse reference genome, EquCab2.0, via a GGGenome search. These procedures can be potentially used to identify therapeutic oligonucleotides, whose nucleotides are unknown, for gene doping control., (© 2021 John Wiley & Sons, Ltd.)

Gene doping is a threat to fair competition in sports, both human and equestrian. One method of gene doping is to administer exogenous genetic materials, called transgenes, into the bodies of postnatal humans and horses. Polymerase chain reaction (PCR)-based transgene detection methods such as digital PCR and real-time PCR have been developed for gene doping testing in humans and horses. However, the significance of PCR inhibitors in gene doping testing has not been well evaluated. In this study, we evaluated the effects of PCR inhibitors on transgene detection using digital PCR and real-time PCR against gene doping. Digital PCR amplification was significantly inhibited by high concentrations of proteinase K (more than 0.1 μg/μl), ethylenediaminetetraacetic acid (more than 5 nmol/μl), and heparin (more than 0.05 unit/μl) but not by ethanol or genomic DNA. In addition, phenol affected droplet formation in the digital PCR amplification process. Real-time PCR amplification was inhibited by high concentrations of phenol (more than 1% v/v), proteinase K (more than 0.001 μg/μl), ethylenediaminetetraacetic acid (more than 1 nmol/μl), heparin (more than 0.005 unit/μl), and genomic DNA (more than 51.9 ng/μl) but not by ethanol. Although both PCR systems were inhibited by nearly the same substances, digital PCR was more robust than real-time PCR against the inhibitors. We believe that our findings are important for the development of better methods for transgene detection and prevention of false negative results in gene doping testing., (© 2021 John Wiley & Sons, Ltd.)

Gene doping is banned in human sports, horseracing, and equestrian sports. One possible form of gene doping is to administer exogenous genes, called transgenes. Several transgene detection methods based on quantitative PCR have been developed. In this study, we investigated the robustness of digital PCR and real-time PCR in transgene detection using primers and probes that matched (P-true) or incompletely matched (P-false) the template DNA. Fluorescence intensity was significantly reduced when substituted probes were used compared to that using the matched probe in both digital and real-time PCR assays. Digital PCR yielded a similar copy number regardless of the probe (P-true: 1230.7, P-false: 1229.7), whereas real-time PCR revealed a decrease in sensitivity based on C q values (P-true: 23.5, P-false: 29.7). When substituted primers were used, the detected copy number decreased in the digital PCR assay, and the C q value in real-time PCR was much higher. Interestingly, digital PCR copy numbers improved by performing PCR at a low annealing temperature, even if a substituted probe was used. Thus, when primer and probe sequences did not completely match the template transgene, digital PCR was relatively robust, but real-time PCR was less sensitive. Although PCR specificity may be reduced, PCR sensitivity can be improved by lowering the annealing temperature. If the target sequence is substituted to escape doping detection, it may be desirable to set the annealing temperature lower and use a more robust method, such as digital PCR, to increase the detection of positive cases, which will also result in fewer false-negative results.

Gene doping has raised concerns in human and equestrian sports and the horseracing industry. There are two possible types of gene doping in the sports and racing industry: (1) administration of a gene-doping substance to postnatal animals and (2) generation of genetically engineered animals by modifying eggs. In this study, we aimed to identify genetically engineered animals by whole-genome resequencing (WGR) for gene-doping control. Transgenic cell lines, in which the erythropoietin gene (EPO) cDNA form was inserted into the genome of horse fibroblasts, were constructed as a model of genetically modified horse. Genome-wide screening of non-targeted transgenes was performed to find structural variation using DELLY based on split-read and paired-end algorithms and Control-FREEC based on read-depth algorithm. We detected the EPO transgene as an intron deletion in the WGR data by the split-read algorithm of DELLY. In addition, single-nucleotide polymorphisms and insertions/deletions artificially introduced in the EPO transgene were identified by WGR. Therefore, genome-wide screening using WGR can contribute to gene-doping control even if the targets are unknown. This is the first study to detect transgenes as intron deletions for gene-doping detection.

Ever since 'One Health' concept was introduced in early 2000s, judicious use of antimicrobials by veterinarians has become an issue of great concern. Recently, findings of anti-inflammatory effects in certain types of antimicrobials have raised a subject for discussion among racing authorities. Regulatory framework of antimicrobials in racing should be based on best interest of horse welfare and doping control perspective, but basic data on prevalence of antimicrobials are lacking. Analysis of 100 postrace urinary samples collected from 10 Japanese racecourses by targeting 21 antimicrobials using ultra performance liquid chromatography-tandem mass spectrometry resulted in detection of ceftiofur, cefalotin, cefalotin metabolite, dihydrostreptomycin, gentamicin, kanamycin, and oxytetracycline. Detection of antimicrobials critically important for resistance in human medicine was limited to a single sample. Oxytetracycline, which is known to possess anti-inflammatory effects, was detected in three samples. This may suggest the need for establishing a regulatory framework from doping control perspective and further studies to clarify pharmacologically relevant concentration of antimicrobials with such properties., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Gene doping, an activity which abuses and misuses gene therapy, is a major concern in sports and horseracing industries. Effective methods capable of detecting and monitoring gene doping are urgently needed. Although several PCR-based methods that detect transgenes have been developed, many of them focus only on a single transgene. However, numerous genes associated with athletic ability may be potential gene-doping material. Here, we developed a detection method that targets multiple transgenes. We targeted 12 genes that may be associated with athletic performance and designed two TaqMan probe/primer sets for each one. A panel of 24 assays was prepared and detected via a microfluidic quantitative PCR (MFQPCR) system using integrated fluidic circuits (IFCs). The limit of detection of the panel was 6.25 copy/μL. Amplification-specificity was validated using several concentrations of reference materials and animal genomic DNA, leading to specific detection. In addition, target-specific detection was successfully achieved in a horse administered 20 mg of the EPO transgene via MFQPCR. Therefore, MFQPCR may be considered a suitable method for multiple-target detection in gene-doping control. To our knowledge, this is the first application of microfluidic qPCR (MFQPCR) for gene-doping control in horseracing.

Objective: Clinical research on gene therapy has advanced the field of veterinary medicine, and gene doping, which is the illegal use of gene therapy, has become a major concern in horseracing. Since the International Federation of Horseracing Authorities defined the administration of oligonucleotides and its analogues as a genetic therapy in 2017, the development of therapeutic nucleotide-detection techniques has become an urgent need. Most currently marketed and developed oligonucleotide therapeutics for humans consist of modified nucleotides to increase stability, and phosphorothioate (PS) modification is common., Results: We demonstrated the specific detection of phosphorothioated oligonucleotides (PSOs) using LC/MS/MS. PSOs produce the specific product ion (m/z 94.9362) derived from PS moiety. PS is not derived from endogenous substances in animal body, and the product ion is a suitable marker for the detection of PSOs. With our strategy, reproducible target analyses were achieved for identifying the specific substances, with a LOD of 0.1 ng/mL and a quantification rage of 0.1-200 ng/mL in deproteinated plasma. Non-target analyses could also detect the presence of PSOs selectively with 100 ng/mL in the same matrix. These results suggested that the detection of PSOs in horse blood is possible by targeting the product ion using LC/MS/MS.

Objective: Doping control is an important and indispensable aspect of fair horse racing; genetic doping has been recently included to this. In this study, we aimed to develop a detection method of gene doping. A plasmid cloned with human erythropoietin gene (p.hEPO, 250 μg/head) was intramuscularly injected into a microminipig. Subsequently, p.hEPO was extracted from 1 mL of plasma and detected by droplet digital polymerase chain reaction., Results: The results confirmed that the maximum amount of plasmid was detected at 15 min after administration and the majority of the plasmid was degraded in the bloodstream within 1-2 days after administration. In contrast, low amounts of p.hEPO were detected at 2-3 weeks after administration. These results suggest that the proposed method to detect gene doping can help obtain information for experiments using horses.