The true stone composition and abnormality of urinary metabolic lithogenic factors of rats fed diets containing melamine

To better understand the toxicity of melamine to humans, the stone composition and urinary metabolic lithogenic factors of rats fed diets containing melamine including the infant’s melamine-induced stone composition were studied. Sixty 4-week-old male rats divided into three groups were, respectively, fed diets containing no melamine (control), 0.1 % melamine, and 1 % melamine for 4 weeks. At the end of experiment, the collected stones and 24-h urines from rats were, respectively, measured with compositions and metabolic lithogenic parameters. The stone from an infant who ingested melamine-adulterated formula was also included in compositional analysis. Across three groups, the stone was only detected in 1 % melamine group, with composition of almost melamine different from the affected infant’s stone composed of melamine and uric acid with a ratio of 1:2. Compared with control group, urine calcium and phosphate excretions were significantly increased in 1 % melamine group. Urine uric acid excretion was significantly increased but citrate excretion was significantly decreased in 0.1?% and 1?% melamine groups. Urine oxalate excretion and pH were indicated without any significant difference. In addition based on urine physicochemical characters, melamine–uric acid stone seems difficult to be formed in the rats due to their characters of urine high-pH and low-uric acid. These results demonstrated that (1) the stone composition of rats fed melamine was not and could not be as that of infants fed melamine-adulterated formula, two species had a different mechanism of melamine-induced stone formation; (2) the exposure of melamine could result in abnormalities of urine metabolic lithogenic factors to rats, perhaps as well as human beings.     

单人经口胆道镜与射频消融术同台诊治不可切除肝外胆管癌的可行性与安全性研究

目的　探讨单人经口胆道镜联合射频消融术（radiofrequency ablation,RFA）同台诊治不可切除肝外胆管癌的可行性和安全性。方法　回顾2013年1月至2022年1月期间在杭州市第一人民医院治疗的90例可疑肝外胆管癌患者资料,根据诊治过程最终纳入69例,分为常规分次组（n=34）：先经内镜逆行胰胆管造影术（endoscopic retrograde cholangiopancreatography, ERCP）+细胞刷或单人经口胆道镜检查取得活检组织,待获得阳性病理结果再次行ERCP+RFA;同台诊治组（n=35）：行ERCP经单人经口胆道镜检查胆道并对病灶行直视下活检,对术中快速病理结果确定为恶性肿瘤的患者同台行RFA。对比两组操作成功率、术后胆红素恢复情况、ERCP次数、术后不良事件发生率、住院时间及费用。结果　两组患者均成功完成内镜下RFA,操作成功率100.0%（69/69）。分次组和同台组总胆红素术后下降50%及以上的患者比例差异无统计学意义［52.94%（18/34）比57.14%（20/35）,χ2=0.27,P=0.604］。分次组ERCP次数明显多于同台组［（2.59±0.50）次/人比（1.00±0.00）次/人］,差异有统计学意义（t=3.13,P=0.002）。分次组和同台组术后总体不良事件发生率差异无统计学意义［（67.65%（23/34）比65.71%（23/35）,χ2=2.83 ,P=0.626］。分次组住院时间明显长于同台组,差异有统计学意义［（17.41±9.13） d比（7.91±3.48） d,t=5.32,P=0.001］。分次组住院费用明显多于同台组,差异有统计学意义［（37 127.88±3 763.77）元比（23 980.69±4 767.15）元,t=6.61,P=0.001］。结论　单人经口胆道镜直视下诊断+活检联合RFA同台诊治不可切除肝外胆管癌可减少ERCP次数,并且不增加术后不良事件发生率,是一种安全有效且具有较高成本-效益比的诊治方法。     

Arthroscopic versus mini-open rotator cuff repair: a prospective,randomized study with 24-month follow-up

This prospective, randomized study was performed to evaluate the results of mini-open and arthroscopic rotator cuff repair in a comparative case series of patients followed for 24 months. A total of 125 patients were randomized to mini-open (Group I) or arthroscopic (Group II) rotator cuff repair at the time of surgical intervention. The University of California Los Angeles (UCLA) score, the American Shoulder and Elbow Surgeons (ASES) index, and muscle strength were measured to evaluate the clinical results, while magnetic resonance arthrography was used at 24-month follow-up to investigate the postoperative rotator cuff integrity. Fifty-three patients in Group I and 55 patients in Group II were available for evaluation at 24-month follow-up. At 24-month follow-up, the UCLA score, the ASES index, and muscle strength were statistically significantly increased in both groups postoperatively, while no significant difference was detected between the 2 groups. Intact rotator cuffs were investigated in 42 patients in Group I and 35 in Group II, and there was a significant difference in postoperative structural integrity between the two groups (P < 0.05). When analysis was limited to the patients with full-thickness tear, the muscle strength of the shoulder was significantly better in Group II, and the retearing rate was significantly higher in Group II. Based on the results obtained from this study, it can be indicated that arthroscopic and mini-open rotator cuff repair displayed substantially equal outcomes, except for higher retearing rate in the arthroscopic repair group. While for patients with full-thickness tear, arthroscopic rotator cuff repair displayed better shoulder strength and significantly higher retearing rate as compared to mini-open rotator cuff repair at 24-month follow-up.     

Analysis of changes in hepatic gene expression in a murine model of tolerance to acetaminophen hepatotoxicity (autoprotection)

Pretreatment of mice with a low hepatotoxic dose of acetaminophen (APAP) results in resistance to a subsequent, higher dose of APAP. This mouse model, termed APAP autoprotection was used here to identify differentially expressed genes and cellular pathways that could contribute to this development of resistance to hepatotoxicity. Male C57BL/6J mice were pretreated with APAP (400 mg/kg) and then challenged 48 h later with 600 mg APAP/kg. Livers were obtained 4 or 24 h later and total hepatic RNA was isolated and hybridized to Affymetrix Mouse Genome MU430_2 GeneChip. Statistically significant genes were determined and gene expression changes were also interrogated using the Causal Reasoning Engine (CRE). Extensive literature review narrowed our focus to methionine adenosyl transferase-1 alpha (MAT1A), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), flavin-containing monooxygenase 3 (Fmo3) and galectin-3 (Lgals3). Down-regulation of MAT1A could lead to decreases in S-adenosylmethionine (SAMe), which is known to protect against APAP toxicity. Nrf2 activation is expected to play a role in protective adaptation. Up-regulation of Lgals3, one of the genes supporting the Nrf2 hypothesis, can lead to suppression of apoptosis and reduced mitochondrial dysfunction. Fmo3 induction suggests the involvement of an enzyme not known to metabolize APAP in the development of tolerance to APAP toxicity. Subsequent quantitative RT-PCR and immunochemical analysis confirmed the differential expression of some of these genes in the APAP autoprotection model. In conclusion, our genomics strategy identified cellular pathways that might further explain the molecular basis for APAP autoprotection.     

A novel fluorometric assay for aldo-keto reductase 1C3 predicts metabolic activation of the nitrogen mustard prodrug PR-104A in human leukaemia cells

Aldo-keto reductase 1C3 (AKR1C3, EC 1.1.1.188) metabolises steroid hormones, prostaglandins and xenobiotics, and activates the dinitrobenzamide mustard prodrug PR-104A by reducing it to hydroxylamine PR-104H. Here, we describe a functional assay for AKR1C3 in cells using the fluorogenic probe coumberone (a substrate for all AKR1C isoforms) in conjunction with a specific inhibitor of AKR1C3, the morpholylurea SN34037. We use this assay to evaluate AKR1C3 activity and PR-104A sensitivity in human leukaemia cells. SN34037-sensitive reduction of coumberone to fluorescent coumberol correlated with AKR1C3 protein expression by immunoblotting in a panel of seven diverse human leukaemia cell lines, and with SN34037-sensitive reduction of PR-104A to PR-104H. SN34037 inhibited aerobic cytotoxicity of PR-104A in high-AKR1C3 TF1 erythroleukaemia cells, but not in low-AKR1C3 Nalm6 pre-B cell acute lymphocytic leukaemia (B-ALL) cells, although variation in PR-104H sensitivity confounded the relationship between AKR1C3 activity and PR-104A sensitivity across the cell line panel. AKR1C3 mRNA expression showed wide variation between leukaemia patients, with consistently higher levels in T-ALL than B-ALL. In short term cultures from patient-derived paediatric ALL xenografts, PR-104A was more potent in T-ALL than B-ALL lines, and PR-104A cytotoxicity was significantly inhibited by SN34037 in T-ALL but not B-ALL. Overall, the results demonstrate that SN34037-sensitive coumberone reduction provides a rapid and specific assay for AKR1C3 activity in cells, with potential utility for identifying PR-104A-responsive leukaemias. However, variations in PR-104H sensitivity indicate the need for additional biomarkers for patient stratification.     

The flavoprotein FOXRED2 reductively activates nitro-chloromethylbenzindolines and other hypoxia-targeting prodrugs

The nitro-chloromethylbenzindoline prodrug SN29428 has been rationally designed to target tumour hypoxia. SN29428 is metabolised to a DNA minor groove alkylator via oxygen-sensitive reductive activation initiated by unknown one-electron reductases. The present study sought to identify reductases capable of activating SN29428 in tumours. Expression of candidate reductases in cell lines was modulated using forced expression and, for P450 (cytochrome) oxidoreductase (POR), by zinc finger nuclease-mediated gene knockout. Affymetrix microarray mRNA expression of flavoreductases was correlated with SN29428 activation in a panel of 23 cancer cell lines. Reductive activation and cytotoxicity of prodrugs were measured using mass spectrometry and antiproliferative assays, respectively. SN29428 activation under hypoxia was strongly attenuated by the pan-flavoprotein inhibitor diphenyliodonium, but less so by knockout of POR suggesting other flavoreductases contribute. Forced expression of 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR), as well as POR, increased activation of SN29428 in hypoxic HCT 116 cells. SN29428 activation strongly correlated with expression of POR and also FAD-dependent oxidoreductase domain containing 2 (FOXRED2), in cancer cell lines. This association persisted after removing the effect of POR enzyme activity using first-order partial correlation. Forced expression of FOXRED2 increased SN29428 activation and cytotoxicity in hypoxic HEK293 cells and also increased activation of hypoxia-targeted prodrugs PR-104A, tirapazamine and SN30000, and increased cytotoxicity of the clinical-stage prodrug TH-302. Thus this study has identified three flavoreductases capable of enzymatically activating SN29428, one of which (FOXRED2) has not previously been implicated in xenobiotic metabolism. These results will inform future development of biomarkers predictive of SN29428 sensitivity.     

Detection of QTc interval prolongation using jacket telemetry in conscious non-human primates: comparison with implanted telemetry

Background and Purpose: During repeat-dose toxicity studies, ECGs are collected from chemically or physically-restrained animals over a short timeframe. This is problematic due to cardiovascular changes caused by manual restraint stress and anesthesia, and limited ECG sampling. These factors confound data interpretation, but may be overcome by using a non-invasive jacket-based ECG collection (JET). The current study investigated whether a jacketed external telemetry system could detect changes in cardiac intervals and heart rate in non-human primates (NHPs), previously implanted with a PCT transmitter.Experimental Approach: Twelve male cynomolgus monkeys were treated weekly with vehicle or sotalol (8, 16, 32 mg kg⁻¹) p.o. ECGs were collected continuously for 24 hours, following treatment, over 4 weeks. A satellite group of six NHPs was used for sotalol toxicokinetics.Key Results: Sotalol attained C_max values 1–3 hours after dosing, and exhibited dose-proportional exposure. In jacketed NHPs, sotalol dose-dependently increased QT/QTc intervals, prolonged PR interval, and reduced heart rate. Significant QTc prolongation of 27, 54 and 76 msec was detected by JET after 8, 16, and 32 mg kg⁻¹ sotalol, respectively, compared with time-matched vehicle-treated animals. Overall, JET-derived PR, QT, QTc intervals, QRS duration, and heart rate correlated well with those derived from PCT.Conclusions and Implications: The current findings clearly support the use of JET to quantify cardiac interval and rhythm changes, capable of detecting QTc prolongation caused by sotalol. JET may be a preferred method compared to restraint-based ECG because high-density ECG sampling can be collected in unstressed conscious monkeys, over several weeks.