Interaction between buprenorphine and norbuprenorphine in neonatal opioid withdrawal syndrome.

Buprenorphine (BUP) is the preferred treatment for opioid use disorder during pregnancy but can cause neonatal opioid withdrawal syndrome (NOWS). Norbuprenorphine (NorBUP), an active metabolite of BUP, is implicated in BUP-associated NOWS. We hypothesized that BUP, a low-efficacy agonist of mu opioid receptors, will not antagonize NorBUP, a high-efficacy agonist of mu opioid receptors, in producing NOWS. To test this hypothesis, we treated pregnant Long-Evans rats with BUP (0, 0.01, 0.1 or 1mg/kg/day) ± NorBUP (1mg/kg/day) from gestation day 9 until pup delivery, and tested pups for opioid dependence using our established NOWS model. We used LC-MS-MS to quantify brain concentrations of BUP, NorBUP, and their glucuronide conjugates. BUP had little effect on NorBUP-induced NOWS, with the exception of 1mg/kg/day BUP significantly increasing NorBUP-induced NOWS by 58% in females. BUP and NorBUP brain concentrations predicted NOWS in multiple linear regression models. Interestingly, NorBUP contributed more to NOWS in females (ßNorBUP = 51.34, p = 0.0001) than in males (ßNorBUP = 19.21, P = 0.093), while BUP was similar for females (ßBUP = 10.62, P = 0.0017) and males (ßBUP = 11.38, P = 0.009). We are the first to report that NorBUP induces NOWS in the presence of BUP and it is more influential in females than males in the contribution of NorBUP to BUP-associated NOWS. These findings suggest that females are more susceptible to NorBUP-induced NOWS, and that treatment strategies that reduce prenatal NorBUP exposure may be more effective for females than males.

Qualitative content analysis of public responses to an FDA inquiry on the impact of scheduling changes to kratom.

BACKGROUND: The legal status of kratom in the United States is complex and varies by state. The U.S. Food and Drug Administration (FDA) and the U.S. Drug Enforcement Administration have repeatedly subjected kratom to regulatory review. However, there hasn't been a systematic review of the public's perception of kratom. The present study analyzed open-ended responses from the public to an FDA solicitation for information regarding kratom with the goal of providing a comprehensive assessment of motives for kratom use. METHODS: To guide decisions regarding kratom regulation, the FDA solicited comments regarding kratom abuse potential, medical usefulness, and impact of scheduling changes from July through August 2021 and posted them to the Federal Register website. We analyzed comments posted during the first 6 weeks of comment solicitation (6,353) using an inductive approach via qualitative content analysis. RESULTS: Respondents reported 106 independent health-related reasons for kratom use, with most categorized as mental health, pain management, substance use disorder, or miscellaneous purposes that included increasing focus, treating insomnia, and decreasing fatigue. Neurological diseases and digestive disorders were also reported. Relatively few (< 2%) responses reported recreational use, abuse potential, or adverse effects of kratom. CONCLUSIONS: Although kratom is not approved as a safe and effective therapy for any indication, individuals use kratom for a broad spectrum of health-related purposes. Limitations of this study include potential bias for respondents with perceived positive experiences using kratom, lack of demographics data, and lack of independent verification of claims made by respondents. Regardless, this study reflects perceptions regarding the therapeutic uses of kratom and provides insight into potential individual-level consequences of regulating kratom in the U.S. It is important to study the public's perception of kratom use, which can aid regulatory purposes and provide clinically important information on individuals' use and valuation of kratom.

Overexpression of MnSOD Protects against Cold Storage-Induced Mitochondrial Injury but Not against OMA1-Dependent OPA1 Proteolytic Processing in Rat Renal Proximal Tubular Cells.

Kidneys from deceased donors undergo cold storage (CS) preservation before transplantation. Although CS is a clinical necessity for extending organ quality preservation, CS causes mitochondrial and renal injury. Specifically, many studies, including our own, have shown that the triggering event of CS-induced renal injury is mitochondrial reactive oxygen species (mROS). Here, we explored the role of OMA1-depedent OPA1 proteolytic processing in rat kidney proximal tubular epithelial (NRK) cells in an in vitro model of renal CS (18 h), followed by rewarming (6 h) (CS + RW). The involvement of mROS was evaluated by stably overexpressing manganese superoxide dismutase (MnSOD), an essential mitochondrial antioxidant enzyme, in NRK cells. Western blots detected rapid OPA1 proteolytic processing and a decrease in ATP-dependent cell viability in NRK cells subjected to CS + RW compared to control cells. Small interfering RNA (siRNA) knockdown of OMA1 reduced proteolytic processing of OPA1, suggesting that OMA1 is responsible for OPA1 proteolytic processing during CS + RW-induced renal injury. Overexpression of MnSOD during CS + RW reduced cell death, mitochondrial respiratory dysfunction, and ATP-dependent cell viability, but it did not prevent OMA1-dependent OPA1 processing. These data show for the first time that OMA1 is responsible for proteolytically cleaving OPA1 in a redox-independent manner during renal cell CS.

Fluorescence-Based Assay For Measuring OMA1 Activity.

Mitochondrial fusion depends on proteolytic processing of the dynamin-related GTPase protein, OPA1, which is regulated by the mitochondrial zinc metalloproteinase, OMA1. Last year we published a report describing a novel approach to directly measure the enzymatic activity of OMA1 in whole cell lysates. This fluorescence-based reporter assay utilizes an eight amino acid peptide sequence referred to as the S1 cleavage site where OMA1 cleaves within OPA1 and is flanked by a fluorophore and quencher. In this chapter, we provide additional insight into the OMA1 activity assay.

The BK activator NS11021 partially protects rat kidneys from cold storage and transplantation-induced mitochondrial and renal injury.

Kidneys from deceased donors used for transplantation are placed in cold storage (CS) solution during the search for a matched recipient. However, CS induces mitochondrial and cellular injury, which exacerbates renal graft dysfunction, highlighting the need for therapeutic interventions. Using an in vitro model of renal CS, we recently reported that pharmacological activation of the mitochondrial BK channel (mitoBK) during CS protected against CS-induced mitochondrial injury and cell death. Here, we used an in vivo syngeneic rat model of renal CS (18 h) followed by transplantation (24 h reperfusion) (CS + Tx) to similarly evaluate whether addition of a mitoBK activator to the CS solution can alleviate CS + Tx-induced renal injury. Western blots detected the pore-forming α subunit of the BK channel in mitochondrial fractions from rat kidneys, and mitoBK protein level was reduced after CS + Tx compared to sham surgery. The addition of the BK activator NS11021 (3 µM) to the CS solution partially protected against CS + Tx-induced mitochondrial respiratory dysfunction, oxidative protein nitration, and cell death, but not acute renal dysfunction (SCr and BUN). In summary, the current preclinical study shows that pharmacologically targeting mitoBK channels during CS may be a promising therapeutic intervention to prevent CS + Tx-induced mitochondrial and renal injury.

The first direct activity assay for the mitochondrial protease OMA1.

Mitochondria continually undergo fission and fusion which allow mitochondria to rapidly change their shape, size, and function throughout the cell life cycle. OMA1, a zinc metalloproteinase enzyme, is a key regulator of the mitochondrial fusion machinery. The paucity of information regarding OMA1 regulation and function largely stems from the fact that there is no direct method to quantitatively measure its activity. Using a fluorescence-based reporter assay, we developed a sensitive method to measure OMA1 enzymatic activity in whole cell lysates.

Modulation of dietary methionine intake elicits potent, yet distinct, anticancer effects on primary versus metastatic tumors.

Methionine dependency describes the characteristic rapid in vitro death of most tumor cells in the absence of methionine. Combining chemotherapy with dietary methionine deprivation [methionine-deficient diet (MDD)] at tolerable levels has vast potential in tumor treatment; however, it is limited by MDD-induced toxicity during extended deprivation. Recent advances in imaging and irradiation delivery have created the field of stereotactic body radiotherapy (SBRT), where fewer large-dose fractions delivered in less time result in increased local-tumor control, which could be maximally synergistic with an MDD short course. Identification of the lowest effective methionine dietary intake not associated with toxicity will further enhance the cancer therapy potential. In this study, we investigated the effects of MDD and methionine-restricted diet (MRD) in primary and metastatic melanoma models in combination with radiotherapy (RT). In vitro, MDD dose-dependently sensitized mouse and human melanoma cell lines to RT. In vivo in mice, MDD substantially potentiated the effects of RT by a significant delay in tumor growth, in comparison with administering MDD or RT alone. The antitumor effects of an MDD/RT approach were due to effects on one-carbon metabolism, resulting in impaired methionine biotransformation via downregulation of Mat2a, which encodes methionine adenosyltransferase 2A. Furthermore, and probably most importantly, MDD and MRD substantially diminished metastatic potential; the antitumor MRD effects were not associated with toxicity to normal tissue. Our findings suggest that modulation of methionine intake holds substantial promise for use with short-course SBRT for cancer treatment.

Mouse population-based evaluation of urinary protein and miRNA biomarker performance associated with cisplatin renal injury.

Discovery and qualification of novel biomarkers with improved specificity and sensitivity for detection of xenobiotic-induced injuries is an area of active research across multiple sectors. However, the majority of efforts in this arena have used genetically limited rodent stocks that lack variability in xenobiotic responses inherent to genetically heterogeneous human populations. In this study, genetically diverse Diversity Outbred (DO) mice were used as a surrogate for human clinical populations to investigate performance of urinary kidney biomarkers against classical preclinical kidney injury biomarkers (blood urea nitrogen [BUN] and serum creatinine). In this study, cisplatin was used as a paradigm kidney toxicant, with female adult DO mice exposed to a single injection (5 mg/kg) of cisplatin or vehicle and necropsied 72 h post-exposure and 18 h following overnight urine collection. Interindividual variability in kidney toxicity was observed, with DO mice experiencing either no tubule cell necrosis or minimal-mild necrosis. A panel of urinary protein biomarkers and profiled miRNAs were assessed by receiver-operating characteristic curves as to their ability to distinguish non-responder versus responder animals, as defined by histopathological evidence of renal tubule cell necrosis. A surprising outcome of these studies was that BUN was elevated alongside of urinary miRNA and protein biomarkers in animals with grade 2 proximal tubular cell necrosis; but not elevated with grade 1 necrosis. These studies demonstrate a novel approach for using a rodent population to better assess sensitivity of candidate biomarkers, especially for proposed clinical applications. Impact statement Recent studies have indicated that several urinary proteins and miRNA species may be suitable as biomarkers for acute kidney injury. A major focus on biomarker qualification is demonstrating improved specificity and sensitivity over gold standard tests. In this study, a mouse population model, Diversity Outbred mice, was used to demonstrate that neither the urinary protein markers nor the miRNA species assayed in urine could reliably detect low severity kidney injury better than blood urea nitrogen. This study has implications for use of these biomarkers in the clinic, where interindividual heterogeneity is present within patient populations and for which the underlying tissue pathology may not be known.

Renal cold storage followed by transplantation impairs expression of key mitochondrial fission and fusion proteins.

BACKGROUND: The majority of transplanted kidneys are procured from deceased donors which all require exposure to cold storage (CS) for successful transplantation. Unfortunately, this CS leads to renal and mitochondrial damage but, specific mitochondrial targets affected by CS remain largely unknown. The goal of this study is to determine whether pathways involved with mitochondrial fusion or fission, are disrupted during renal CS. METHODS: Male Lewis rat kidneys were exposed to cold storage (CS) alone or cold storage combined with transplantation (CS/Tx). To compare effects induced by CS, kidney transplantation without CS exposure (autotransplantation; ATx) was also used. Mitochondrial function was assessed using high resolution respirometry. Expression of mitochondrial fusion and fission proteins were monitored using Western blot analysis. RESULTS: CS alone (no Tx) reduced respiratory complex I and II activities along with reduced expression of the primary mitochondrial fission protein, dynamin related protein (DRP1), induced loss of the long form of Optic Atrophy Protein (OPA1), and altered the mitochondrial protease, OMA1, which regulates OPA1 processing. CS followed by Tx (CS/Tx) reduced complex I, II, and III activities, and induced a profound loss of the long and short forms of OPA1, mitofusin 1 (MFN1), and mitofusin 2 (MFN2) which all control mitochondrial fusion. In addition, expression of DRP1, along with its primary receptor protein, mitochondrial fission factor (MFF), were also reduced after CS/Tx. Interestingly, CS/Tx lead to aberrant higher molecular weight OMA1 aggregate expression. CONCLUSIONS: Our results suggest that CS appears to involve activation of the OMA1, which could be a key player in proteolysis of the fusion and fission protein machinery following transplantation. These findings raise the possibility that impaired mitochondrial fission and fusion may be unrecognized contributors to CS induced mitochondrial injury and compromised renal graft function after transplantation.

One-carbon metabolism and ionizing radiation: a multifaceted interaction.

Ionizing radiation (IR) is a ubiquitous component of our environment and an important tool in research and medical treatment. At the same time, IR is a potent genotoxic and epigenotoxic stressor, exposure to which may lead to negative health outcomes. While the genotoxocity is well described and characterized, the epigenetic effects of exposure to IR and their mechanisms remain under-investigated. In this conceptual review, we propose the IR-induced changes to one-carbon metabolism as prerequisites to alterations in the cellular epigenome. We also provide evidence from both experimental and clinical studies describing the interactions between IR and one-carbon metabolism. We further discuss the potential for the manipulation of the one-carbon metabolism in clinical applications for the purpose of normal tissue protection and for increasing the radiosensitivity of cancerous cells.

Combination histamine and serotonin treatment after simulated childbirth injury improves stress urinary incontinence.

AIMS: Histamine and serotonin-related pharmaceuticals have the potential to modulate micturition and continence. The aim of this study was to determine if treatment with histamine and/or serotonin improves stress urinary incontinence (SUI) in female rats. METHODS: Twenty-six age-matched female rats underwent pudendal nerve crush and vaginal distension (PNC + VD), to produce SUI. One week after injury, rats were treated subcutaneously with saline, histamine (1.1 µg), serotonin (2µg), or the combination of both twice daily for another week. A sham injured group received sham PNC + VD and were treated with saline (n = 7). Leak point pressure (LPP) testing with simultaneous external urethral sphincter (EUS) electromyography (EMG) was conducted 2 weeks after injury. The urethra was harvested for qualitative and quantitative histology. Data were analyzed with a one-way ANOVA and Student-Newman-Keuls posthoc test with P < 0.05 indicating statistically significant differences between groups. RESULTS: Combination treatment significantly increased LPP after PNC + VD compared to injured sham treatment and treatment with either histamine or serotonin alone. Compared to injured sham treated rats, all three treatments significantly increased EUS EMG amplitude at both baseline and peak pressure and EUS EMG firing rate at peak pressure during LPP testing. There were more consistent urethral striated muscle fibers and thicker smooth and striated muscle with combination and histamine treatment. There was a statistically significant shift to a greater proportion of thicker collagen fibers in the urethra in serotonin and combination treated rats compared with injured sham treated rats. CONCLUSIONS: Combination treatment was the most effective and may provide an effective therapy for SUI. Neurourol. Urodynam. 35:703-710, 2016. © 2015 Wiley Periodicals, Inc.