Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells.

Reactive oxygen species formed within the mammalian cell can produce 8-oxo-7,8-dihydroguanine (8-oxoG) in mRNA, which can cause base mispairing during gene expression. Here we found that administration of 8-oxoGTP in MTH1-knockdown cells results in increased 8-oxoG content in mRNA. Under this condition, an amber mutation of the reporter luciferase is suppressed. Using second-generation sequencing techniques, we found that U-to-G changes at preassigned sites of the luciferase transcript increased when 8-oxoGTP was supplied. In addition, an increased level of 8-oxoG content in RNA induced the accumulation of aggregable amyloid ß peptides in cells expressing amyloid precursor protein. Our findings indicate that 8-oxoG accumulation in mRNA can alter protein synthesis in mammalian cells. Further work is required to assess the significance of these findings under normal physiological conditions.

Characterization of a novel CYP2C9 mutation (1009C>A) detected in a warfarin-sensitive patient.

Warfarin is the most frequently prescribed anticoagulant for the long-term treatment in the clinic. Recent studies have shown that polymorphic alleles within the CYP2C9, VKORC1, and CYP4F2 genes are related to the warfarin dosage requirement. In this study, a novel non-synonymous mutation (1009C>A) in CYP2C9 was detected in a warfarin-hypersensitive patient, while the other two candidate genes were both found to be homozygous for the wild-type alleles. The newly identified point mutation results in an amino acid substitution at position 337 of the CYP2C9 protein (P337T) and has been designated as the novel allele CYP2C9*58. When expressed in insect cell microsomes, the relative intrinsic clearance values of the CYP2C9.58 variant for tolbutamide and losartan were quite similar to those of the typical defective variant CYP2C9.3, whereas the clearance value of CYP2C9.58 for diclofenac was slightly higher than that of another typical defective variant CYP2C9.2. These data suggested that when compared with wild-type CYP2C9.1, the enzymatic activity of the novel allelic variant has been greatly reduced by the 1009C>A mutation. If patients carrying this allele take drugs metabolized by CYP2C9, their metabolic rate might be slower than that of wild-type allele carriers and thus much more attention should be paid to their clinical care.

Effects of estradiol and methoxychlor on Leydig cell regeneration in the adult rat testis.

The objective of the present study is to determine whether methoxychlor (MXC) exposure in adulthood affects rat Leydig cell regeneration and to compare its effects with estradiol (E2). Adult 90-day-old male Sprague-Dawley rats received ethane dimethane sulfonate (EDS) to eliminate the adult Leydig cell population. Subsequently, rats were randomly assigned to four groups and gavaged with corn oil (control), 0.25 mg/kg E2 and 10 or 100 mg/kg MXC daily from days 5 to 30 post-EDS treatment. The results showed that MXC and E2 reduced serum testosterone levels on day 58 post-EDS treatment. qPCR showed Hsd17b3 mRNA levels were downregulated 7-15 fold by E2 and MXC, indicating that development of the new population of Leydig cells was arrested at the earlier stage. This observation was supported by the results of histochemical staining, which demonstrated that Leydig cells in MXC-treated testis on day 58 post-EDS treatment were mostly progenitor Leydig cells. However, Pdgfb mRNA levels were downregulated, while Lif transcript levels were increased by MXC. In contrast, E2 did not affect gene expression for these growth factors. In conclusion, our findings indicated that both MXC and E2 delayed rat Leydig cell regeneration in the EDS-treated model, presumably acting by different mechanisms.

Simultaneous measurement of acalabrutinib, ibrutinib, and their metabolites in beagle dog plasma by UPLC-MS/MS and its application to a pharmacokinetic study.

In our present experiment, the aim of this paper was to develop and fully validate an accurate and simple ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay for simultaneous quantification of acalabrutinib, ibrutinib, and their metabolites (ACP-5862 and PCI-45227) in beagle dog plasma and to survey the pharmacokinetic study of all analytes in beagle dogs. After a quick protein precipitation with acetonitrile, the chromatographic separation of acalabrutinib, ACP-5862, ibrutinib, PCI-45227 and bosutinib (internal standard, IS) were finished on an Acquity BEH C18 (2.1 mm × 50 mm, 1.7 µm) column and their detections were conducted by a Xevo TQ-S triple quadrupole tandem mass spectrometer in the positive ion mode. The assay displayed excellent linearity in the calibration range of 0.5-300 ng/mL for acalabrutinib, 0.5-30 ng/mL for ACP-5862, and 0.5-200 ng/mL for both ibrutinib and PCI-45227, respectively. Notably, the lower limit of quantification (LLOQ) for each compound was determined to be 0.5 ng/mL. The accuracy of all analytes for intra- and inter-day ranged from -4.3% to 14.6%, while precision were ≤9.0%. The recovery of each substance was > 81.0%, and no significant matrix effects were observed. The stabilities of all analytes under different conditions met all requirements for the quantitation in plasma samples. In addition, our developed UPLC-MS/MS method could also be employed to measure the pharmacokinetic profiles of acalabrutinib, ACP-5862, ibrutinib, and PCI-45227 in beagle dog plasma.

Development of UPLC-MS/MS method for studying the pharmacokinetic interactions of pexidartinib with antifungal drugs in rats.

Pexidartinib was approved in the USA for targeted therapy of adult patients with symptomatic tenosynovial giant cell tumour (TGCT) by the FDA. The purpose of our study was to develop and establish a quick assay based on ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for the measurement of pexidartinib concentrations in plasma and to survey whether antifungal drugs (isavuconazole, posaconazole, fluconazole and itraconazole) could change the pharmacokinetic parameters of pexidartinib in rats. After the quick protein crash with acetonitrile, the chromatographic separation of pexidartinib and upadacitinib (used as the internal standard in this study, IS) were conducted on an Acquity BEH C18 (2.1 × 50 mm, 1.7 µm) column, and the detection of the analyte was also accomplished with a Xevo TQ-S triple quadrupole tandem mass spectrometer in the positive ion electro-spray ionization (ESI) interface. The assay showed good linearity in the range of 1-7000 ng/mL. The accuracy and precision were all within the acceptable limits in the bioanalytical method, and the results of recovery, matrix effect, stability, and carry-over were also met the requirements. The application of the validated UPLC-MS/MS bioanalytical method was further successfully involved in the drug-drug interactions study from rats. It was found that fluconazole and itraconazole significantly increased the concentration of pexidartinib and had the inhibitory effect on the metabolism of pexidartinib, while not isavuconazole and posaconazole. Thus, more attention should be paid to the concurrent use of pexidartinib with fluconazole or itraconazole to reduce the risk of unexpected clinical outcomes.

Oxidative DNA and RNA damage and their prognostic values during Salmonella enteritidis-induced intestinal infection in rats.

Emerging evidence suggests that microbial pathogens may induce oxidative stress in infected hosts. The aim of the present study was to investigate the relationship between changes in oxidative stress and intestinal infection with and without antibiotic treatment in animal models. Sprague-Dawley (SD) rats were divided into three groups: rats infected with Salmonella enterica serovar Enteritidis (S. enteritidis), rats infected with S. enteritidis followed by norfloxacin treatment, and the control group. To evaluate oxidative stress changes, levels of 8-oxo-7,8-dihydroguanosine (8-oxo-Gsn) and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxo-dGsn), which represented oxidative damage to RNA and DNA, respectively, were analysed in urine and tissue samples. In urine, the level of 8-oxo-Gsn increased significantly after oral exposure to S. enteritidis (p ≤ 0.001) and returned to baseline after recovery. Notably, norfloxacin treatment decreased the level of 8-oxo-Gsn in urine significantly (p = 0.001). Changes of 8-oxo-Gsn measured in tissues from the small intestine, colon, liver and spleen were consistent with 8-oxo-Gsn measured in urine. Our study suggested that 8-oxo-Gsn in urine may serve as a highly sensitive biomarker for evaluating the severity of S. enteritidis infection and the effectiveness of antibiotic treatment against infection.

Determination and pharmacokinetic study of pirfenidone in rat plasma by UPLC-MS/MS.

A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was developed and validated for the determination and pharmacokinetic investigation of pirfenidone in rat plasma. Sample preparation was accomplished through a simple one-step deproteinization procedure with 0.2 mL of acetonitrile to a 0.1 mL plasma sample. Plasma samples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consisting of acetonitrile-0.1% formic acid in water with gradient elution. The total run time was 3.0 min and the elution of pirfenidone was at 1.39 min. The detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m/z 186.2→92.1 for pirfenidone and m/z 237.1→194.2 for carbamazepine (IS), respectively. The calibration curve was linear over the range of 5-2000 ng/mL with a lower limit of quantitation (LLOQ) of 5 ng/mL. Mean recovery of pirfenidone in plasma was in the range of 80.4-84.3%. Intra-day and inter-day precision were both <12.1%. This method was successfully applied in pharmacokinetic study after oral administration of 10.0mg/kg pirfenidone in rats.