bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatocyte‐specific YAP deletion suppresses hepatocyte proliferation and hepatomegaly induced by CAR agonist, TCPOBOP (1,4‐Bis [2‐(3,5‐Dichloropyridyloxy)] benzene), in mice
Authors: Bhushan Bharat , Banerjee Swati , Stoops John W. , Mars Wendy M. , Orr Anne , Bowen William C. , Paranjpe Shirish , Michalopoulos George K.
Published: June 2021

TCPOBOP (1,4‐Bis [2‐(3,5‐Dichloropyridyloxy)] benzene) is a direct ligand of constitutive androstane receptor (CAR), which induces robust hepatocyte proliferation and hepatomegaly in mice without any liver injury or tissue loss. Organ size including that of liver is known to be tightly controlled by transcriptional regulator, yes‐associated protein (YAP). Recent studies have indicated potential interaction of CAR and YAP. Here, we directly investigated role of YAP in TCPOBOP‐induced proliferative response using hepatocyte‐specific YAP‐KO strategy. Hepatocyte‐specific YAP deletion was achieved by administering AAV8‐TBG‐CRE vector to YAP‐floxed mice. TCBOPOP treatment in control mice caused rapid activation and nuclear translocation of YAP along with decreased phosphorylation (inactive form) of YAP concomitant to proliferative response. YAP deletion neither altered protein expression of CAR nor its activation. However, YAP deletion significantly reduced TCPOBOP‐induced hepatomegaly and hepatocyte proliferation. TCPOBOP‐driven cell cycle activation was disrupted in YAP‐KO mice due to impaired protein and gene expression of cyclin D1 and decreased phosphorylation of retinoblastoma (Rb) protein. YAP regulated these cell cycle proteins via alerting expression of cMyc and FOXM1, two critical transcriptional regulators of TCPOBOP‐mediated hepatocyte proliferation. In conclusion, our study revealed an important role of YAP signaling in hepatomegaly and hepatocyte proliferation induced by TCPOBOP.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Determining the opioid sensitivity of projections from the Kölliker‐Fuse to respiratory rhythm generators
Authors: Bateman Jordan T , Levitt Erica S
Published: June 2021

Opioid‐induced respiratory suppression is the primary cause of death in individuals who overdose on opioids, but the mechanism remains elusive. Mu opioid receptor agonists have been shown to hyperpolarize a population of neurons in the Kölliker‐Fuse nucleus (KF) due to activation of G protein‐mediated inwardly rectifying (GIRK) conductance (Levitt et al., J Physiol, 2015). The KF, required for normal eupneic breathing, adjusts respiratory rhythm and rate by modulating respiratory rhythm generators. Given the interconnectivity of the KF and respiratory rhythm generators, we hypothesize that KF neurons projecting to respiratory rhythm generators are opioid‐sensitive and thus a possible mechanism by which opioids reduce respiratory rate. The goal of these experiments was to determine if opioid‐sensitive KF neurons project to respiratory rhythm generators. Fluorescent beads (FluoSpheres 580/605, ThermoFisher) were injected into respiratory rhythm generators, including pre‐Bötzinger complex (preBötC) and post‐inspiratory complex (PiCo), of mice. Whole‐cell voltage‐clamp recordings were made from retrograde‐labeled neurons in the KF. Mu opioid receptor‐mediated outward currents were identified in KF neurons that project to the preBötC. Therefore, opioid‐sensitive connections between the KF and respiratory rhythm generators may be involved in opioid‐induced respiratory suppression.

Support or Funding Information

Supported by NIH grant DA038069.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Sex Differences in Behavioral Strategies are Accompanied by Differences in Accumbal Dopamine Release
Authors: Johnson Amy , Brady Lillian , Thibeault Kimberly , Lopez Alberto , Kutlu Munir Gunes , Calipari Erin
Published: June 2021

While preclinical work has aimed to outline the neural mechanisms of drug addiction, it has overwhelmingly focused on male subjects. There has been a push in recent years to incorporate females into existing addiction models; however, males and females often have different behavioral strategies, making it important to not only include females, but to develop models that assess the factors that comprise female drug addiction. Although both males and females become addicted to cocaine, females transition to addiction faster, consume more cocaine, and have greater difficulty remaining abstinent. Clinical work shows that women also exhibit increased cue‐induced craving for cocaine, indicating that sex differences in cocaine addiction may not solely be due to reinforcing effects of cocaine and may include complicated interactions with drug‐associated stimuli. Here we define the circuit‐based mechanisms by which females exhibit enhanced vulnerability to cocaine addiction. By combining fast‐scan cyclic voltammetry, optogenetics, and drug self‐administration we link pathway‐specific dopamine signaling to the motivational state of females over the estrous cycle. We find that female mice are more motivated to take cocaine, and are more responsive to cocaine‐associated cues. Female mice also respond differently than males for optical stimulation of the ventral tegmental area to nucleus accumbens dopamine neurons. We link these changes to differences in dopamine release as well as the relationship between releasable pools and available calcium over the estrous cycle. These data suggest that several factors could be playing a role in sex differences in addiction and provide a mechanism by which the estrous cycle can modulate dopamine release which may produce long‐lasting effects on cues, cue learning, and drug‐taking behavior.

Support or Funding Information

Funding was provided by startup funds from Vanderbilt University School of Medicine Department of Pharmacology (E.S.C), as well as funding from the National Institutes of Health (NIH). Funds from the National Institute of Drug Abuse (NIDA) DA042111(E.S.C), National Insitute of Mental Health (NIMH) MH064913 (K.C.T) & MH065215‐15 (A.R.J), the Brain and Behavior Research Foundation (to E.S.C), Whitehall Foundation (to E.S.C), Edward J Mallinckrodt Jr. Foundation (to E.S.C) also supported this work.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Activation of TAAR1 attenuates extended access of cocaine self‐administration and stress‐induced reinstatement of cocaine‐seeking
Authors: Liu Jian‐Feng , Johnson Bernard , Seaman Robert , Wu Ruyan , Zhang Yanan , Li Jun‐Xu
Published: June 2021

Trace amine‐associated receptor 1 (TAAR1) is the best‐characterized sub‐family of receptors of trace amines. As a modulator of dopaminergic system, TAAR1 has been shown to play a critical role in regulating the rewarding properties of drugs of abuse such as cocaine. In our previous studies, we demonstrated that TAAR1 agonists were able to reduce cocaine intake and cue‐ and drug‐induced reinstatements of cocaine‐seeking under short‐access conditions. However, compared to the extended access of cocaine self‐administration model, the short‐access model could not mimic some core properties of addiction, such as escalation, compulsive motivation, and incubation of cocaine‐seeking. Furthermore, it remains unclear whether activation of TAAR1 would affect stress‐induced reinstatement of cocaine‐seeking. Here, we investigated the effects of TAAR1 partial agonist RO5263397 on the extended access of cocaine self‐administration. We also assessed the effects of the selective TAAR1 full agonist RO5166017 on the yohimbine‐induced reinstatement of cocaine‐seeking. We found that the TAAR1 partial agonist RO5263397 attenuated the escalation, break point, and cue‐seeking behavior in the extended access of cocaine self‐administration model. We also found that RO5166017 reduced yohimbine‐induced reinstatement of cocaine‐seeking and yohimbine‐potentiated cue‐induced reinstatement of cocaine seeking, indicating that activation of TAAR1 decreased the stress‐induced reinstatement of cocaine‐seeking. Taken together, our results suggest that TAAR1 is a promising therapeutic target for the treatment of cocaine addiction.

Support or Funding Information

This work was supported by the National Institute of Health Grants R21‐DA033426.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effects of Response‐Contingent and Non‐Contingent Drug History on the Development of High Levels of MDPV Self‐Administration
Authors: Doyle Michelle R , DeSantis Rachel E , Sulima Agnieszka , Rice Kenner C , Collins Gregory T
Published: June 2021

A subset of rats that self‐administer synthetic cathinones, such as 3,4‐methylenedioxypyrovalerone (MDPV), develop high levels of drug intake and compulsive‐like patterns of responding that may be related to the compulsive, binge‐like patterns of cathinone (e.g., “bath salts”) use reported in humans. Once this “high‐responder” phenotype is established, it transfers to other stimulants; however, a prior history of responding for cocaine, but not food, has been shown to prevent or delay the development of this phenotype. These findings suggested that cocaine produces neuroplastic changes that can prevent or delay the transition to high levels of MDPV intake. The present studies aimed to evaluate whether 1) response contingent and non‐contingent cocaine histories are equally effective at preventing high levels of MDPV intake; and 2) response contingent and non‐contingent MDPV histories are sufficient to establish the high‐responder phenotype in rats self‐administering cocaine. Four groups of male Sprague‐Dawley rats were assigned to initially receive MDPV or cocaine in a response contingent or non‐contingent manner using a “yoked” self‐administration paradigm under a fixed ratio (FR) 1 schedule (i.e., response contingent drug infusions delivered to rat A were paired with non‐contingent infusions of the same drug to rat B). After 20 sessions of this yoked MDPV or cocaine self‐administration procedure (10 sessions at FR1, 10 sessions at FR5), all rats were allowed to self‐administer the alternate drug under an FR5 schedule for an additional 20 sessions (e.g. both cocaine groups now self‐administered MDPV). Subsequently, full FR5 dose response curves were generated by dose substitution. These studies found that, unlike response‐contingent cocaine delivery, non‐contingent cocaine is unable to prevent the development of high levels of MDPV self‐administration. Additionally, non‐contingent MDPV is not sufficient to establish high levels of cocaine intake. These results suggest that the contingencies surrounding drug delivery are important for both establishing and preventing the high‐responder phenotype, indicating that both a behavioral and pharmacological history may be important for the transition from regulated to compulsive patterns of drug intake.

Support or Funding Information

This work was supported by NIH/NIDA grant (R01 DA039146; GTC), NIDA‐ and NIAAA‐IRPs (KCR), and NIH Predoctoral Training Program in the Neurosciences (T32 NS082145; MRD).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacokinetic, Cardiovascular and Locomotor Effects of alpha‐Pyrrolidinopentiophenone (alpha‐PVP) in Male Sprague Dawley Rats after Active Vaccination
Authors: McClenahan Samantha J , Kormos Chad M , Mu Shengyu , Hambuchen Michael D , Carroll Ivy F , Owens S Michael
Published: June 2021

Alpha‐pyrrolidinopentiophenone (alpha‐PVP) is a psychoactive synthetic cathinone that elicits pharmacological effects similar to methamphetamine and cocaine and at high doses can produce psychosis, cardiovascular toxicity, and death. The goal for these studies was to characterize the pharmacokinetics (PK) of alpha‐PVP and test an active vaccine for treating alpha‐PVP‐induced effects. The hapten‐protein conjugate vaccine was developed from an alpha‐PVP/MDPV‐like hapten structure capable of generating high affinity antibodies that significantly cross react with both alpha‐PVP and 3,4‐methylenedioxypyrovalerone (MDPV). Previous studies showed the bi‐specific vaccine could significantly reduce locomotor effects of MDPV. For the current studies, we hypothesized that treatment with this vaccine could stimulate production of high affinity antibodies against alpha‐PVP capable of altering the PK disposition and attenuating the cardiovascular and locomotor effects of alpha‐PVP. The hapten was conjugated to immunocyanin monomers of keyhole limpet hemocyanin, mixed with Sigma Adjuvant System, and used to immunize male Sprague Dawley rats (n=6/group) at 0, 3, and 9 weeks. Pharmacological testing occurred between 12–26 weeks from the start of immunizations. For the PK studies, three doses of alpha‐PVP (0.56, 1, and 3 mg/kg) were administered subcutaneously (sc) to control and vaccinated rats (n=6/group/dose); and tail vein blood samples were collected over time. PK values of alpha‐PVP showed linear PK with a half‐life of 1.7 h in controls and a significant (p<0.05) treatment‐dependent difference in the area under the alpha‐PVP concentration time curve between control and vaccinated rats. In a separate group of rats (n=5–6/group), cardiovascular parameters recorded by radiotelemetry showed a significantly lower area under the heart rate and blood pressure time curves after 0.56, 1 and 3 mg/kg alpha‐PVP in vaccinated rats compared to controls. Finally, locomotor studies of control and vaccinated rats (n=6/group) were conducted using a series of increasing doses of alpha‐PVP (0.3–5.6 mg/kg, sc, 2 days apart). The immunized rats displayed significantly lower alpha‐PVP‐induced locomotor activity and a significantly shorter duration of action compared to controls. In conclusion, alpha‐PVP has linear PK in control rats, and active vaccination with a bi‐specific conjugate vaccine significantly increased the binding of alpha‐PVP in the serum and reduced alpha‐PVP‐induced cardiovascular and locomotor effects compared to controls.

Support or Funding Information

Funded by NIDA grants DA039195 and F31 DA046121, and T32 GM106999

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Locomotor Effects of Substituted Amphetamines and Cathinones in Mice: Structure‐Activity Relationships
Authors: Russell Lauren , Fukuda Saki , Hyatt William , Berquist Michael , Landavazo Antonio , Blough Bruce , Fantegrossi William
Published: June 2021

Substituted amphetamines represent one of the largest and most pharmacologically‐diverse classes of abused drugs. Modifications to the amphetamine base structure are seemingly limitless, and novel psychoactive substituted amphetamines continuously emerge on the illicit market. Amphetamine analogues containing a ketone at the beta carbon are referred to as cathinones, which are typically the active constituents in abused “bath salts” products. Each “generation” of cathinone analogues introduces new structural motifs, most likely in response to regulatory control of previous compounds. One recently identified “bath salts” constituent is 4‐chloroethcathinone (4‐CEC), consisting of the base amphetamine structure with the addition of an ethyl side chain, a chlorine at the 4‐position, and a ketone group at the beta carbon. In these studies, we compared 12 structurally‐analogous substituted amphetamines and cathinones increasing in structural complexity from amphetamine to 4‐CEC in order to assess structure‐activity relationships for locomotor stimulant and toxic effects in mice. Automated photobeam chambers were used to record various aspects of mouse locomotor activity including jump count, time in stereotypy, vertical time, speed, and distance. Full dose‐effect curves were determined for each compound, starting with a dose that produced saline‐like distance traveled, and increasing in half‐log units until distance decreased from that drug's peak, or until lethal effects were observed. For the substituted amphetamines, increasing the length of the side chain increased maximal effectiveness on locomotor endpoints. As compared to analogous unsubstituted compounds, addition of a chlorine at the 4‐position did not substantially impact distance traveled, but increased time spent in stereotypy. These same trends were also observed with the substituted cathinones. Interestingly, 4‐CEC produced lethality in all subjects at doses on the ascending limb of the dose‐effect curve for distance traveled. Further development of structure‐activity relationships for these drugs may determine which moieties of their chemical structures are involved in producing specific pharmacological effects, and may allow predictions about abuse liability or toxic effects of new pharmacological entities with similar structural motifs.

Support or Funding Information

These studies supported by DA022981, R01‐DA039195, and DEA/FDA HHSF223201610079C.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Serotonin 2A and Sigma 1 Receptor Antagonists Synergize to Block the Acute Toxicity of Methamphetamine
Authors: Ray Azizi
Published: June 2021

Currently there are no Food and Drug Administration approved pharmacotherapies for methamphetamine (METH) overdose, thus identifying novel drug targets to prevent this devastating adverse event is a public‐health imperative. Previous research suggests that serotonin and sigma receptors may contribute to the adverse effects of METH.

Determine whether antagonism of serotonin 2A (5‐HT2A) and sigma 1 (σ1) alone or in combination attenuates METH‐induced lethality, hyperthermia, convulsions, and seizures.

Male, Swiss Webster mice received intraperitoneal injections of the selective 5‐HT2A receptor antagonist M100907 (1–10 mg/kg), the selective σ1 receptor antagonist BD 1047 (10–30 mg/kg), and a combination of M100907/BD 1047 (1:10 mg/kg) prior to treatment with METH (78 mg/kg ‐ LD67). Convulsions and lethality were assessed by observation, core body temperature was assessed by surgically implanted telemetric probes, and seizures were assessed by electroencephalography.

M100907 and BD 1047 administered alone had no significant effect on METH‐induced lethality. However, combined antagonism of 5‐HT2A and σ1 receptors eliminated lethality in all mice tested (a reduction from LD67 to LD0). Similarly, only the combination of 5‐HT2A and σ1 receptor antagonism eliminated seizure activity. All three treatments decreased tonic‐clonic convulsions as measured on a modified Racine scale. None of the treatments decreased METH‐induced hyperthermia.

This research suggests that reducing METH‐induced seizures may be critical for reducing METH‐induced lethality. These data support the continued investigation of the role of 5‐HT2A and σ1 receptors in METH overdose, including their potential to yield emergency reversal agents.

Support or Funding Information

These studies were supported by the National Institutes on Drug Abuse [NS100512] and by funding from the Mercer University College of Pharmacy (Murnane KS‐ Principal Investigator; Canal CE– Principal Investigator)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
To Study the Correlation of Vitamin D and Metabolic Syndrome in Schizophrenia
Authors: Ambwani Sneha R , Nebhinani Naresh , Sharma Praveen , Misra Arup Kumar , Saini Vijay Kumar , Sharma Pramod Kumar , Dwivedi Pradeep
Published: June 2021

Objectives

Schizophrenia, a highly heterogeneous & biological disorder of the brain, is associated with biochemical, metabolic & cardiovascular derangements. Vitamin D is known to have the role as a neuro‐modulator/neuroprotection in the brain. Prevalence of metabolic syndrome in schizophrenia is very high & it further increases with the use of atypical antipsychotic drugs. The present study is conducted to correlates the association between serum levels of vitamin D and parameters of metabolic syndrome in the patient of schizophrenia.

Methods

Venous blood sample was collected from 50 adult schizophrenic patients. Serum vitamin D and parameters of metabolic syndrome like waist circumference, blood pressure (systolic as well as diastolic), fasting blood glucose (FBS), triglycerides (TG) and high‐density lipoprotein (HDL) was measured. An analysis was done using the SPSS version 20.0 for Windows (Chicago, Illinois, USA). Mean and the standard deviation is calculated for the continuous variables. Pearson's correlation was used to assess the relationship between vitamin D level and parameters of metabolic syndrome.

Results

Systolic blood pressure (SBP), diastolic blood pressure (DBP), fasting blood glucose (FBS) and triglycerides (TG) were found to have an inverse relation with serum vitamin D level. Vitamin D level was found in the deficient category (<20 ng/dl) in most of the patients. Waist Circumference and high‐density lipoprotein (HDL) was found to have a direct correlation with vitamin D in the patient of schizophrenia of the population.

Conclusions

The study showed the possible clinical role of vitamin D level in initiating changes in the biochemical and physical parameters of metabolic syndrome in patients of schizophrenia even though the sample size is small. It might signify the role of Vitamin D in the pathogenesis of metabolic syndrome in schizophrenia.

Support or Funding Information

The present study is a part of my intramural project funded by All India Institute of Medical Sciences, Jodhpur, Rajasthan, India

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Allostatic plasticity of cAMP system drives opioid induced adaptations in striatal dopamine signaling
Authors: Muntean Brian S , Dao Maria T , Martemyanov Kirill A
Published: June 2021

Opioids generate euphoria by activating the reward system in the brain, however chronic exposure generates plastic changes in signal processing to program aversive reactions. A core element of the reward circuit involves the integration of dopamine in subpopulations of medium spiny striatal neurons, D1‐MSN and D2‐MSN, whereby these two parallel channels encode the opponent processes through cAMP‐dependent signaling. However, the precise mechanisms that bias circuit activity to encode a range of hedonic states during various stages of opioid exposure are not well understood. Uncovering the fundamental properties and molecular machinery for parsing GPCR signal integration in striatal neurons is a critical step in advancing our understanding of neurobiology necessary to comprehend the adaptations induced by opioids. By recording cAMP dynamics with nanomolar resolution from genetically defined striatal neurons in an intact neuronal circuit, we revealed elemental properties that promote D1‐MSN signaling during acute opioid exposure while favoring D2‐MSN signaling following chronic drug administration. Additionally, we identified two key molecular components that shape plasticity of cAMP signaling. We found that opioids enable phosphodiesterases to regulate D1‐MSN signaling capacity while reducing the role of presynaptic dopamine transporters to influence D2‐MSN response strength. Our results demonstrate the importance of balancing signal integration between concurrently activated subpopulations of striatal neurons while exploring pre‐ and postsynaptic mechanisms that impact accompanying computations performed by the circuit. We propose a quantitative model whereby differential exposure to opioids provides cell‐specific adjustments in cAMP signal strength that imbalances circuit activity to promote transitions between states of reward and aversion.

Support or Funding Information

This work was supported by NIH grants: DA041207 (to B.S.M.), DA036596 (to K.A.M.), and DA026405 (to K.A.M.)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
HISTOPATHOLOGICAL EFFECTS OF A GLUTAMINE‐FREE DIET IN A MOUSE MODEL OF ASTROCYTOPATHY (ALEXANDER DISEASE)
Authors: PALOMEQUE JESUS CARLOS GARCIA , CHAMIZO ANDRES MOLERO , BAÑARES ROSA LEON
Published: June 2021

The aim of this study was to explore the histopathological effects of a glutamic acid and glutamine‐free diet (the Glutamic deficient Diet U8958V243) in mice with a selective mutation (R76H, R236H) for the glial fibrillary acidic protein gene (GFAP). This diet was administered for 5 months after weaning. Four groups were included in the study: (i) mice with GFAP active and standard diet (n = 6); mice with GFAP active and glutamic deficient diet (n = 6); mice without GFAP active and standard diet (n =6); mice without GFAP active and glutamic free diet (n =6). Hematoxylin‐eosin and Klüver‐Barrier procedures were used for histological analysis. The histopathological differences between groups were analyzed by unifactorial ANOVA and t‐test post‐hoc comparisons. Different degrees of histopathological changes, including demyelination, Rosenthal fibers formation, modification in oligodendrocytes, and accumulation of astrocytes, were found in the groups of transgenic mice, in areas typically affected in astrocytopathy models. These results show for the first time the effect of the control of aminoacids in the diet on the histopathology of mouse models of Alexander’ disease.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Druggability evaluation of a protamine‐like peptide
Authors: Li Tong , Dou Guifang
Published: June 2021

Background

Protamine (Protamine sulfate, PS) is a highly cationic arginine‐rich peptide isolated from sperm of the salmonidae or clupeidae family. PS which is widely used as an UFH antidote. PS, which possessed good efficacy, however, has many adverse effects including systematic hypotension, anaphylactic reaction, anaphylactoid reactions and catastrophic pulmonary hypertension by the virtue of its heterogeneous and highly alkaline nature.

Purpose

To prepare and evaluate an effective and low toxic heparin antagonistic drug.

Methods

Stability of R15S samples in room temperature during 30 days was measured using High performance liquid chromatograph (HPLC). Comparisons on UFH neutralization ability, between PS and R15S, were made aided by active partial thromboplastin time (APTT), anti‐Xa and anti‐IIa assays in vitro and in vivo. For the drug toxicity, MTT tests and acute toxicity assays.

Results

The R15 sulfate, R15S, was prepared in our lab and its stability for 30 days in room temperaturewas good. The results of APTT, anti‐Xa assays and anti‐IIa assays in vitro and in vivo showed that R15S has equal heparin neutralizing ability to PS. There was no cytotoxicity for R15S and PS at the concentration of 60μg·mg‐1 or below, but R15S showed lower cytotoxicity than PS at 250 μg·mg‐1 to1000 μg·mg‐1.

Conclusion

We prepared and evaluated a new UFH antagonist, R15S. R15S showed compatable UFH reversal ability, similar drug toxicity and superior safety in immunology, which made it a potential candidate deserving further studies.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of Potent Pyrazole‐Based Biased Small Molecule APJ Receptor Agonists
Authors: Narayanan Sanju , Vasukuttan Vineetha , Rajagopal Sudarshan , Maitra Rangan , Runyon Scott P
Published: June 2021

The apelinergic system comprises the APJ receptor and its cognate apelin and ELABELA peptides of various lengths. This system has become an increasingly attractive target for cardiometabolic diseases. However, in vivo studies of this receptor are difficult because endogenous peptides are labile with short half‐lives and small molecule ligands are under reported. Recently, we discovered a novel pyrazole based small molecule agonist of APJ (EC50 = 21.5 μM, Ki = 5.2 μM) through focused screening of a compound library, which was further optimized to our initial lead (EC50 = 800 nM, Ki = 1.3 μM). In our efforts to synthesize more potent agonists and to explore the structural features important for APJ agonism, we carried out structural modifications at the pyrazole core as well as the amino acid side‐chain of our initial lead. Systematic modifications at these two positions provided potent small molecule agonists exhibiting EC50 values of ~100 nM using separate calcium mobilization and cAMP generation functional assays. Recruitment of β‐arrestin as a measure of desensitization potential of select compounds was also investigated. Functional selectivity was a feature of several compounds with a bias towards calcium mobilization over β‐arrestin recruitment. These compounds may be suitable for chronic in vivo studies of APJ and await additional characterization.

Support or Funding Information

NIH Grants: 1R01HD079547‐01A1 and 1R01DK103625‐01A1

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structure‐based design and in vivo anti‐arthritic activity evaluation of a potent cyclopropyl peptidyl nitrile inhibitor of cathepsin C
Authors: Korkmaz Brice , Lesner Adam , Gieldon Artur , Lauritzen Conni , Pedersen John
Published: June 2021

Cathepsin C (CatC) is a dipeptidyl‐exopeptidase which activates neutrophil serine protease precursors (elastase, proteinase 3, cathepsin G and NSP4) by removing their N‐terminal propeptide in bone marrow cells at the promyelocytic stage of neutrophil differentiation. The resulting active neutrophil serine proteases are implicated in chronic inflammatory and autoimmune diseases. Hence, inhibition of CatC represents a therapeutic strategy to suppress excessive neutrophil serine protease activities in various neutrophil mediated diseases. We designed and synthesized a series of peptidyl cyclopropyl nitrile compounds as putative CatC inhibitors. One of these compounds, IcatC ((S)‐2‐amino‐N‐((1R,2R)‐1‐cyano‐2‐(4′‐(4‐methylpiperazin‐1‐ylsulfonyl)biphenyl‐4‐yl)cyclopro‐pyl)butanamide)) was identified as a cell‐permeable and potent inhibitor of both human and rodent CatC. Pharmacokinetic analysis in mice showed that IcatC was retained in the bone marrow and reached sufficient tissue levels for CatC inhibition. Subcutaneous administration of IcatC in a mouse model of rheumatoid arthritis induced by monoclonal anti‐collagen antibodies exhibited statistically significant anti‐arthritic activity, with sustained reductions in mean total arthritis scores, mean rear paw arthritis scores and mean rear paw thickness.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of antioxidants and biotransformation inhibitors on 1,2,3‐trichlorobenzene nephrotoxicity in vitro
Authors: Rankin Gary O'Neal , Harvey Brandon , Nguyen Vy , Hicks Mason , Anestis Dianne , Leader Tori , Dial Mason , Brown Kathleen , Valentovic Monica
Published: June 2021

Chlorinated benzenes are used as chemical intermediates in the manufacture of a wide range of commercial products and are environmental contaminants in soil and waste water. Toxicity induced by chlorobenzenes includes nephrotoxicity with in vivo nephrotoxicity associated with rat α2u‐globulin. A recent study in our laboratory demonstrated that trichlorobenzenes (TCBs) are directly toxic to the kidney, with 1,2,3‐TCB being the most potent nephrotoxicant in isolated renal cortical cells (IRCC) from male Fischer 344 rats. The purpose of this study was to evaluate several antioxidants and inhibitors of biotransformation pathways to determine their impact on 1,2,3‐TCB nephrotoxicity. IRCC (~4 million cells/ml; 3 ml) were incubated with shaking at 37°C under a 95% oxygen/5% carbon dioxide atmosphere with 1,2,3‐TCB (1.0 mM) or vehicle (dimethyl sulfoxide) for 60 min with or without a pretreatment. General cytotoxicity was determined by assessing trypan blue exclusion of IRCC, measuring changes in lactate dehydrogenase (LDH) release, and quantitating ATP levels. 1,2,3‐TCB cytotoxicity was reduced by all antioxidants (ascorbate, glutathione, alpha‐tocopherol) and general cytochrome P450 (CYP) inhibitors (metyrapone and piperonyl butoxide), but not by peroxidase inhibitors (mercaptosuccinate, indomethacin). Several selective CYP inhibitors (isoniazid, omeprazole, sulfaphenazole, oleandomycin, thiotepa) were tested, and all provided some protection against 1,2,3‐TCB cytotoxicity. Protective pretreatments also generally attenuated 1,2,3‐TCB‐induced reductions in ATP levels. These results suggest that free radicals/oxidative stress and CYP‐mediated biotransformation play a role in 1,2,3‐TCB nephrotoxicity in vitro.

Support or Funding Information

Supported in part by NIH grant P20GM103434.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Differential Nephrotoxicity of Three Brominated Flame Retardants in Rat and Human Renal Cells
Authors: Cummings Brian S , Barnett Lillie M , Siddiquee Naomi M
Published: June 2021

Brominated flame retardants (BFRs) are a class of organohalogens commonly added to commercial products such as computers, electronics, textiles, and furniture to reduce their flammability. BFRs have significant environmental persistence and are reported to be detected in human blood and breastmilk. In particular, tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD), and polybrominated diphenyl ethers (PBDEs) occupy nearly 20 percent of the global flame retardant market and have been reported to have adverse effects on humans and wildlife. As such, the mechanisms of BFR‐induced toxicity are actively being explored under the U.S. EPA Toxic Substance Control Act. Previous research shows that these compounds cause nephrotoxicity in rats and mice; however, the mechanisms mediating this nephrotoxicity are unknown. In the present study, we determined the effects of TBBPA, HBCD, and BDE 47 on cell viability in rat (NRK), human embryonic (HEK‐293), and adult human (HK‐2) kidney cells after 48 hours. We observed a concentration‐ and species‐dependent effect on MTT staining, with IC50 values of 58, 38, and 3 μM for TBBPA; 20, 16, and 7 μM for HBCD; and 46, 40, and 14 μM for BDE 47 in NRK, HEK‐293, and HK‐2 cells, respectively. We assessed the mechanisms of cell death by measuring annexin V staining and propidium iodide (PI) staining as markers of apoptosis and necrosis, and by studying changes in nuclear morphology via DAPI staining. Significant increases in both the percentage of annexin V‐positive cells and the percentage of annexin V and PI double‐positive cells suggested that all compounds were inducing apoptosis. These data suggest that BFRs induce species‐dependent toxicity in renal cells, with higher levels of toxicity being induced in human cells. The mechanisms mediating this differential toxicity is the subject of future studies.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Hepatoprotective Properties of Aqueous and Ethanolic Stem Bark Extracts of Hura crepitans Linn. Against CCl4− Induced Acute Liver Damage in Rats
Authors: Owojuyigbe Oluwole Sunday , Larbie Christopher
Published: June 2021

Medicinal plants are gaining wide acceptance even amongst the literate population of the world. Secondary metabolites obtained from herbal sources contain bioactive phytochemicals, many of which have little or no side effects. The hepatoprotective properties of Hura crepitans against CCl4‐induced acute hepatoxicity were investigated, using 48 male Sprague‐Dawley rats. Liver damage was induced with administration of a 1:1 (v/v) CCl4 and olive oil after pre‐treating for 7 days with ethanolic stem bark extract (EES) and aqueous stem bark extract (AES). Hepatoprotective effects of Hura crepitans were investigated by estimating the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and total bilirubin levels (TBIL). Effects of Hura crepitanson biomarkers of lipid peroxidation (oxidative damage) and antioxidant enzymes, superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S‐transferase (GST) and the non‐enzymic antioxidant, reduced glutathione (GSH) were determined in liver post mitochondrial fraction (PMF). EES, AES and Silymarin displayed significant hepatoprotective activities by reducing the activities of ALT, AST and ALP on one hand, and also decreased the level of TBIL on the other hand. Pre‐treatment with Hura crepitans extracts and the standard drug Silymarin led to reduction in the activities of antioxidant enzymes, reduction in levels of malondialdehyde (MDA) and reduction in serum levels of GSH in a significant manner. The histopathological studies also demonstrate the protective effect of pre‐treatment with Hura crepitans and Silymarin against oxidative damage by CCl4. Our investigations show that Hura crepitans extracts possess significant hepatoprotective effect against CCl4‐induced hepatoxicity, an endowment arising from the constituent phytochemicals. The present study has demonstrated that EES and AES protected the liver from the detrimental effects of CCl4 through their effects on the antioxidant defence system and their abilities to mitigate against lipid peroxidation.

Support or Funding Information

Tertiary Education Trust Fund (TETFund), Nigeria/ASPET.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of tobacco‐specific nitrosamine (TSNA) content in smokeless tobacco products in Bangladesh
Authors: Nasrin Shamema , Chen Gang , Watson Christy J , Lazarus Philip
Published: June 2021

Head and neck cancers are among the most common cancers in Southeast Asia and are likely to face sharp increases in the coming years due to the widespread use of smokeless tobacco (SLT) products. Bangladesh has the second highest oral cancer mortality rate as well as the second highest rate of SLT product usage worldwide. SLT products contain high levels of the highly carcinogenic tobacco‐specific nitrosamines (TSNAs); they include N‐nitrosonornicotine (NNN), 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK), N‐nitrosoanatabine (NAT), and N‐nitrosoanabasine (NAB). To date, the amount of TSNAs present in Bangladeshi SLT products has not been reported. The goal of the present study was to develop an efficient TSNA and nicotine extraction method to quantify the levels of TSNAs and nicotine present in 31 Bangladeshi SLT products. The concentration of NNN, NNK, NAT, and NAB in Bangladeshi SLT brands ranged from 1.07–59.18 μg/g SLT, 0.15–33.70 μg/g SLT, 0.79–44.64 μg/g SLT, and 0.03–12.85 μg/g SLT, respectively. Pan Masala, which is labeled as a non‐tobacco product, also contains low levels of TSNAs. Betel nut, a common flavor additive in SLT products in Bangladesh, contained no detectible TSNA levels. Bangladeshi SLT products contained significantly higher (p<0.05) levels of TSNAs as compared to SLT brands from the United States (U.S.), and were higher on average than the levels observed in brands in other regions of southern Asia. NNN and NNK levels were 16 and 19, 2 and 3.5, and 62 and 85 fold higher than U.S., Indian and Pakistani SLT brands, respectively. Additionally, the levels of NNN and NNK measured in U.S. brands in this study were 1.2‐fold higher than previously reported values from 2013. This study demonstrates that the levels of TSNAs, including the major tobacco carcinogens NNN and NNK, are far higher in Bangladeshi SLT brands than they are in other regions of the world. Exposure to such high levels of carcinogens may be the most important contributing factor to the rate of head and neck as well as other tobacco‐related cancers observed in Bangladesh.

Support or Funding Information

This work is supported in part by grants from NIH, National Cancer Institute (grant R01‐ES025460, to P. Lazarus) and the Health Sciences and Services Authority of Spokane, Washington (grant WSU002292, to College of Pharmacy and Pharmaceutical Sciences, Washington State University)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxidative Stability and Antiproliferative Effect of Fish Oil Commercial Supplements on Cultured Human Keratinocytes
Authors: de Oliveira Carvalho Patricia , de Santis Gabrielle Kristine Doratiotto , Franco Yollanda Edwirges Moreira , Barba Fatima Cristine Ribeiro , de Souza dos Santos Gisele Karine , Longato Giovanna Barbarini
Published: June 2021

Fish oil, due high concentration of n‐3 polyunsaturated fatty acids (n‐3 PUFA), reduce symptoms in many inflammatory skin diseases, limiting the spreading of the inflammatory process. Oxidized derivative was shown to play an important role in the antiproliferative properties of n‐3 PUFA. The objective of this study was to assess the PUFA content, oxidative stability and antiproliferative effects on cultured human keratinocytes of fish oil commercial supplements. Lipid oxidation was measured by colorimetric reactions and the fatty acid composition was measured using gas chromatography. Primary (peroxide), secondary (anisidine), and total oxidation products levels of two supplements exceeded maximum established by international standards of quality. In vitro inhibitory activity against HaCat cell lines, measured with the tetrazolium MTT (3‐(4,5‐dimethylthiazolyl‐2)‐2,5‐diphenyltetrazolium bromide) assay, showed that the supplement of higher oxidation content exerted the most potent antiproliferative effect (IG50 86.54 μg/mL). Our data provide additional information regarding the preferential antiproliferative effect of oxidized derivatives of n‐3 PUFA in cultured human keratinocytes

Support or Funding Information

FAPESP and CAPES (Finance Code 001)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional Characterization of Novel CYP2C9 Variants Found in an Alaska Native Population
Authors: Henderson Lindsay M , McDonald Matthew G , Rettie Allan E , Thummel Kenneth E
Published: June 2021

Coding‐region variants of CYP2C9 can dramatically influence the pharmacokinetics and drug response of therapeutic agents, an important clinical consideration for commonly used CYP2C9 narrow therapeutic index substrates, including (S)‐warfarin and phenytoin. In the Yup'ik Alaska Native people, novel CYP2C9 protein variants M1L, N218I, and P279T are expressed with higher frequencies than the well‐characterized CYP2C9*2 and CYP2C9*3 alleles. This study's objective was to determine the relative expression of these CYP2C9 variants in HepG2 cells and to characterize the function of the purified reconstituted enzymes expressed in E. coli, towards several CYP2C9 drug substrates.

While mRNA expression of the CYP2C9 M1L, N218I, and P279T gene variants and the reference (wildtype) protein in HepG2 cells were similar, the protein was undetectable for the M1L variant. This is expected, due to disruption of the start codon in CYP2C9 M1L. His‐tagged CYP2C9 reference protein and N218I and P279T variants expressed well in E. coli and were highly purified after affinity chromatography. Following reconstitution with cytochrome P450 reductase and cytochrome b5, the N218I and P279T variants metabolized (S)‐warfarin, but not (R)‐warfarin, to the principal 7‐ and 6‐hydroxy metabolites formed by the reference enzyme. Kinetic studies of (S)‐warfarin metabolism demonstrate that the N218I variant has reduced function, with less than 40% of the catalytic efficiency of reference enzyme, while the P279T variant had similar catalytic efficiency to that of reference CYP2C9. Similar, although not identical concentration–rate profiles were obtained for phenytoin, flurbiprofen and naproxen metabolism.

The potential of CYP2C9 M1L, N218I, and perhaps even P279T alleles to alter the pharmacokinetics of drugs metabolized by CYP2C9 may put carriers at risk of exacerbated therapeutic effects from drugs that rely predominantly on CYP2C9 for their metabolic clearance. Further clinical implications for the Alaska Native people include phenotypic misclassification from currently available pharmacogenetic tests, which typically screen only for the common CYP2C9*2 (R144C) and CYP2C9*3 (I359T) variants.

Support or Funding Information

P01 GM116691, T32 GM 007750

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nanoparticle‐Mediated Inhibition of Acute Leukemic Stem Cells
Authors: Glass Sterling , Gonzalez‐Fajardo Laura , Lu Xiuling
Published: June 2021

Chemoresistance accounts for 90% of failed chemotherapeutic treatments. Chemoresistant cancer stem cells (CSC) contribute to these poor outcomes via self‐renewal and non‐tumorigenic differentiation. While CSC subpopulations have been shown to contribute to cancer advancement and recurrence, targeting them has proven difficult. Doxorubicin (DOX) is a potent chemotherapeutic agent used to treat many types of solid and hematologic cancers, including leukemia. Leukemic stem cells (LSC) are associated with chemoresistance, tumor relapse, and poor patient survival. DOX exhibits broad cytotoxicity but is accompanied by severe and sometimes life‐threatening toxicities. We have developed DOX‐loaded polymeric nanoparticles (DOX‐NP) to overcome these problems. These novel, patented nanoparticle delivery systems can diminish adverse effects of DOX administration, such as cardiac toxicity, via improved biodistribution and preferential accumulation in cancer sites. Nanoparticle‐mediated delivery of DOX shows promise as a potential treatment to specifically target LSC. Previous findings from a collaborative effort revealed the ability of low‐dose DOX to specifically inhibit pS552‐β‐catenin, a key mediator of LSC self‐renewal in T‐cell acute lymphocytic leukemia (T‐ALL). Our DOX‐NPs exhibited long circulation times and improved cardiac toxicity compared to free DOX. In vivo, our DOX‐NPs successfully inhibited LSC and promoted normal HSPC recovery with 3‐fold greater median survival than Doxil® (FDA‐approved liposomal DOX) in a patient‐derived xenograft (PDX) model of T‐ALL. In contrast, Doxil® promoted LSC expansion. To identify the mechanisms underlying the DOX‐NP treatment advantages, DOX‐NPs were compared with Doxil® based on in vitro DOX release, cytotoxicity to leukemic cells and bone marrow cells, and in vivo bone marrow accumulation. The DOX‐NPs exhibited slower drug release than Doxil®, which may allow for longer exposure of DOX to LSC. Significant bone marrow accumulation has been observed in both nanoparticle systems. Further understanding of the contribution of DOX accumulation in bone marrow to LSC inhibition in addition to determining the cytotoxicity of both DOX formulations to LSC and leukemic cells will aid in designing an effective drug‐loaded nanotherapeutic to target pS552‐β‐catenin for inhibiting LSC self‐renewal.

Support or Funding Information

This work was supported by the UCONN Academic Plan Grant, the Promising Project Award from the Program in Innovation Therapeutics for Connecticut's Health (PITCH), and the UCONN START PPOC grant.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.