bluuebunny/crossref_metadata_2025 huggingface dataset:
MUCOSAL PROTECTIVE AGENTS PREVENT EXACERBATION OF NSAID‐INDUCED SMALL INTESTINAL LESIONS CAUSED BY ANTISECRETORY DRUGS IN RATS
Authors: Satoh Hiroshi , Amagase Kikuko , Yokoi Ami , Ohashi Masato , Kuwata Sayumi , Mochizuki Sari , Takeuchi Koji
Published: June 2021

Non‐steroidal anti‐inflammatory drugs (NSAIDs) have been reported to be a common cause of small intestinal lesions in humans; however, there are few effective agents for the treatment of this complication. Antisecretory drugs such as histamine H2‐ receptor antagonists and proton pump inhibitors are commonly used for the treatment of upper gastrointestinal mucosal lesions induced by NSAIDs. However, it has recently been reported that these drugs aggravate NSAID‐induced small intestinal lesions in rats. We examined the effect of mucosal protective agents (MPA) on the exacerbation of NSAID‐induced intestinal lesions caused by ranitidine (RAN) or omeprazole (OPZ). Male Wistar rats (180–220g) were used. Both RAN (30 mg/kg) and OPZ (100) significantly increased the intestinal lesions induced by low doses of DIC (3 and 6). All of misoprostol (0.03–0.3), irsogladine (1–10) and rebamipide (10–300) prevented the increase of lesions dose‐dependently. These results indicate that: 1) antisecretory drugs enhance the potential ulcerogenic effect of low (clinical) doses of NSAIDs; and 2) MPAs prevent the exacerbation of NSAID‐induced intestinal lesions caused by antisecretory drugs.
Electrophysiological properties of colon‐projecting sensory neurons in male and female serotonin transporter knockout (SERT KO) rats
Authors: Schneider Stephen , Wang Hui , Fried David , Galligan James
Published: June 2021

Irritable bowel syndrome (IBS) is a gut motility disorder and pain disorder associated with altered serotonin signaling, particularly in women. We used whole cell recordings of spinal sensory neurons in vitro from SERT KO and wild‐type (WT) rats to determine if sex‐related alterations in neuron excitability could contribute to gut pain. Neurons studied were <30–45 microns in diameter. There were no differences in resting membrane potential or input resistance of neurons from WT or SERT KO rats of either sex. The rheobase of neurons from female SERT KO rats was higher than neurons from male WT and SERT KO rats. Female SERT KO neurons when depolarized fired more action potentials than neurons from female WT rats. The variance in neuronal firing rate in female SERT KO neurons was greater than female WT neurons. Action potential firing was not different in neurons from male SERT KO and WT rats. There was no difference in the proportion of neurons with TTX sensitive and resistant action potentials in WT and SERT KO rats of either sex. Colon projecting sensory neurons SERT KO female rats exhibited elevated action potential firing without a change in membrane potential or input resistance. Increased responsiveness of colon‐projecting sensory neurons to membrane depolarization may contribute to visceral hypersensitivity in female IBS patients. (Support: R21NS075841)
The human host defense peptide cathelicidin induces AIF‐ and EndoG‐mediated cell death in colon cancer cells
Authors: CHO CHI HIN , Ren S.X.
Published: June 2021

Human cathelicidin (LL‐37) is implicated in cancer development. In this study, we demonstrate that LL‐37 was substantially downregulated in primary tumor tissues of 58 out of 60 cases of colon cancer as revealed by immunohistochemistry. Functional characterization indicates that LL‐37 reduced cell viability and induced apoptosis in cultured colon cancer cells as evidenced by increased phosphatidylserine externalization and DNA fragmentation. The pro‐apoptotic action of LL‐37 was caspase‐independent as neither LL‐37 induced caspase activation nor its action could be blocked by a pan‐caspase inhibitor. LL‐37‐induced apoptosis was accompanied by the nuclear translocation of apoptosis‐inducing factor (AIF) and endonuclease G (EndoG), knockdown of either of which reversed the pro‐apoptotic action of LL‐37. These molecular events were preceded by p53 activation and p53‐dependent upregulation of Bax and Bak and downregulation of Bcl‐2 in LL‐37‐treated colon cancer cells, in which knockdown or genetic ablation of Bax, Bak or p53 or overexpression of Bcl‐2 abolished LL‐37‐induced nuclear translocation of AIF and EndoG and apoptosis. The pro‐apoptotic effect of LL‐37 was mediated by a pertussis toxin‐sensitive G‐protein‐coupled receptor. Taken together, LL‐37 activates the p53‐Bax/Bak axis to stimulate AIF‐ and EndoG‐mediated caspase‐independent apoptosis in colon cancer cells. These findings may shed light on the novel tumor suppressive function of LL‐37 in colon cancer.
Cathelicidin is a host defense peptide in controlling Helicobacter pylori survival and infection
Authors: ZHANG Lin , Yu Jun , Wu William K.K. , Cho C.H.
Published: June 2021

Cathelicidin, a host defense antibacterial peptide in humans can eradicate different kinds of microbial infection. This study sought to elucidate the actions of cathelicidin in protection against Helicobacter pylori (H. pylori) infection both in vitro and in vivo. To examine the direct antimicrobial action of cathelicidin, H. pylori survival, biofilm formation and morphology change were determined after exposure to different doses of cathelicidin in vitro. Intracellular H. pylori in gastric epithelial cells and in cathelicidin wild‐type (Cnlp+/+) and knockout (Cnlp−/−) mice stomachs were also investigated. Results showed that exogenous cathelicidin could affect H. pylori growth, destroy bacteria biofilm and cause pore formation in H. pylori membranes. Endogenous cathelicidin in gastric epithelial cells could be induced by active form of vitamin D (1,25D3). We also showed that 1,25D3 eliminated the intracellular H. pylori in vitro. Furthermore, Cnlp−/−mice exhibited stronger H. pylori colonization in both acute and chronic H. pylori infection when compared with Cnlp+/+ mice. To further confirm this antibacterial action was due to endogenous cathelicidin, a high level of mouse gastric cathelicidin (CRAMP) production was found in Cnlp+/+ mice but not in Cnlp−/− animals. This finding could partially explain why there was less bacterial infection in wild type mice. Taken together, these results indicate that human cathelicidin plays a significant role as a host defense peptide for H. pylori clearance and infection.
The mechanism of the Ric‐8 protein requirement in heterotrimeric G protein biosynthesis
Authors: Chan PuiYee , Tall Gregory
Published: June 2021

We recently demonstrated newly made Gα subunits are defective in initial association with endomembranes in Ric‐8A−/− cells. To define the precise events by which Ric‐8 mediates Gα biosynthesis, we utilized the rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE) cell‐free translation systems to study potential Ric‐8A influence of Gα subunit translation kinetics and protein folding. Gα subunit translation rates and overall produced protein amounts were equivalent in the Ric‐8A‐ and mock‐immunodepleted lysates. The function of Ric‐8A in Gα biosynthesis was revealed when folded Gα protein levels were quantified. GDP‐AlF4−‐bound Gα subunits produced in mock‐depleted RRL were resistance to limited trypsinolysis, indicating folded Gα subunits. However, Gα subunits produced from Ric‐ 8A‐depleted RRL were not protected. Add back of recombinant Ric‐8A protein to the Ric‐8A‐depleted RRL markedly enhanced protection to trypsin of GDP‐AlF4−‐bound Gα subunits. Similar results were obtained in WGE that has no endogenous Ric‐8 component. WGE‐translated Gαq was resolved by gel filtration and found to be aggregated. Interestingly, Ric‐8A addition resulted Gαq to elute as a ~100 kDa species, and this was dissociable with GTPγS, producing functional Gαq‐GTPγS monomer. Data will be presented showing the concerted action of Ric‐8A with other cellular chaperones in the process of G protein alpha subunit folding.
Activation of the G(s) heterotrimer monitored in living cells with novel G(s) biosensors
Authors: Yano Hideaki , Provasi Davide , Filizola Marta , Ferre Sergi , Javitch Jonathan A
Published: June 2021

Our understanding of conformational changes associated with receptor activation has been advanced profoundly by the crystal structure of agonist‐bound β2 adrenergic receptor (β2AR) coupled to G(s). However, there is uncertainty regarding the extent to which conformational changes in G protein are conserved in living cells as well as across different receptors.

We set out to understand the movement of the G(s) heterotrimer after activation of β2AR in intact cells. Using bioluminescence resonance energy transfer, we made observations on movement within the G protein and between the receptor and the G protein. Using a library of novel G(s) biosensors with either luciferase or GFP inserted at various positions throughout the structure, we studied conformational changes in cells and compared the results to the crystal structures of the inactive and active conformations. We also studied conformational changes in G(s) protein induced by the D1 dopamine receptor and A2A adenosine receptor.

Our analysis using these G(s) biosensors suggests that conformational changes within the G(s) heterotrimer are largely conserved among different G(s)‐coupling receptors. Using this set of G(s) biosensors, partiality of agonists can be studied in relation to structural changes and subsequent effector activation. Thus, these biosensors represent a novel pharmacological tool to study structure‐function relationships.
Induction of CXCR7 Switches SDF‐1 Signaling and Phagocytic Function in Macrophages: a Potential Role in Atherosclerosis
Authors: Ma Wanshu , Liu Yiwei , Ellison Nicholas , Shen Jianzhong
Published: June 2021

Objective

The discovery of CXCR7 as a new receptor for SDF‐1 places many previously described SDF‐1 functions attributed to CXCR4 in question, though whether CXCR7 acts as a signaling or “decoy” receptor has been in debate. It is known that CXCR7 is not expressed in normal blood leukocytes; however, the potential role of leukocyte CXCR7 in disease states has not been addressed.

Methods and Results

We show that CXCR7 was detected in macrophage‐positive area of aortic atheroma of ApoE‐null mice, but not in healthy aorta. During monocyte‐to‐macrophage differentiation, CXCR7 was up‐regulated at mRNA and protein levels, with more expression in M1 than in M2 phenotype. In addition, CXCR7 induction was associated with a SDF‐1 signaling switch from pro‐survival ERK and AKT pathways in monocytes to pro‐inflammatory JNK and p38 pathways in macrophages. The latter effect was mimicked by a CXCR7‐selective agonist TC14012 and abolished by siRNA knockdown of CXCR7. Furthermore, CXCR7 activation increased macrophage phagocytic activity, which was suppressed by CXCR7 siRNA silencing or by inhibiting either the JNK or p38 pathways.

Conclusions

CXCR7 is induced during monocyte‐to‐macrophage differentiation, which is required for SDF‐1 signaling to JNK and p38 pathways, leading to enhanced macrophage phagocytosis, thus possibly contributing to atherogenesis.
Receptor mediated signaling by lysophosphatidic acid and epidermal growth factor in a colon cancer cell line
Authors: Bell Aimee Kristina , Zhang Zhihong , Meier Kathryn
Published: June 2021

Lysophosphatidic acid (LPA) is a lipid mediator that promotes proliferation, migration and survival in many types of cancer cells. LPA binds to G protein‐coupled receptors (LPARs). LPA has a well established, but not well understood, relationship with epidermal growth factor (EGF), a peptide growth factor that binds to tyrosine kinase receptors (EGFRs). Our laboratory has shown that bidirectional cross‐talk can occur between LPARs and EGFRs. A better understanding of the interaction between these two receptors is necessary to develop improved anti‐cancer therapeutics. This study addresses the relationship in a colon cancer model. Both LPA and EGF receptors are known to be overexpressed in colon cancer. In the current study, we use Caco‐2 cells, a human source colorectal adenocarcinoma line that shares brush border characteristics with small intestine tissue. The hypothesis addressed was that cross‐talk between LPARs and EGFRs substantially contributes to overall responses to EGF. Serum‐starved Caco‐2 cells were incubated with and without pertussis toxin to block LPA response. Cells were then treated with fetal bovine serum (10%), LPA (10μM), or EGF (100 nM). Whole‐cell extracts were immunoblotted for activated signaling proteins to determine whether inhibition of LPAR attenuates EGFR activation. The results show that serum, LPA, and EGF all activate Erk, p70S6K, and Akt in Caco‐2 cells. Pertussis toxin reduces protein kinase activation in response to all three agonists. These results suggest that Caco‐2 cells are an appropriate model system in which to explore cross‐talk between EGFRs and LPARs.
A Comparison of the Subjective and Cardiovascular Effects Produced by Exposure to Intravenous versus Smoked Methamphetamine in the Laboratory
Authors: Mahoney James Joseph , Haile Colin , Newton Thomas , Shapiro Ben , De La Garza Richard
Published: June 2021

Previous research has demonstrated that intravenous (IV) and smoked cocaine administration produced similar increases in heart rate (HR) and blood pressure (BP), whereas comparable doses of smoked cocaine resulted in higher subjective ratings of stimulated and high. To our knowledge, there have not been any studies conducted directly comparing the subjective and cardiovascular responses produced by IV methamphetamine (METH) versus smoked METH. Thirteen non‐treatment seeking METH‐dependent individuals, participated in separate protocols in which they were administered IV METH and smoked METH. In the first study, participants were administered METH (0 and 30 mg, IV) and subjective effects, as well as cardiovascular effects, were assessed prior to and 5, 15, 30, 45, and 60 min after METH administration. In the second study, smoked METH (0 and 30 mg) was administered and subjective and cardiovascular effects were obtained on the same time course as above. While not reaching statistical significance, METH administered via the IV route resulted in higher BP and HR across the time course assessed. Interestingly, subjective reports of desire METH and likely to use METH was higher when the route of administration was smoked. These findings demonstrate that the administration of IV versus smoked METH have differential cardiovascular and subjective effects in METH‐dependent participants.
Modulation of the discriminative‐stimulus effects of cocaine by dopamine β‐hydroxlyase inhibitors in rats
Authors: Manvich Daniel F , DeBrouse Lauren , Weinshenker David
Published: June 2021

Inhibitors of dopamine β‐hydroxylase (DBH), the enzyme that converts dopamine (DA) to norepinephrine (NE) in noradrenergic cells, have shown promise for the treatment of cocaine dependence, yet their effects on the interoceptive‐stimulus effects of cocaine are not well characterized. The current experiments examined the impact of DBH inhibitors on the discriminative‐stimulus effects of cocaine. Sprague‐Dawley rats (n=8) were trained to discriminate 5.6 mg/kg cocaine from saline using a multi‐component, food‐reinforced discrimination procedure. On test days, subjects were pretreated with the nonselective DBH inhibitor disulfiram (0 – 100.0 mg/kg i.p.) or the selective DBH inhibitor nepicastat (0 – 56.0 mg/kg i.p.) 2 hr prior to a session in which cocaine (0 – 5.6 mg/kg) was cumulatively administered across components. Both disulfiram and nepicastat failed to generalize to cocaine when tested up to doses that significantly reduced responding. However, in combination studies, both disulfiram and nepicastat produced dose‐dependent leftward shifts in the cocaine dose‐response function. These results suggest that pharmacological inhibition of DBH does not produce cocaine‐like interoceptive‐stimulus effects alone, but functionally enhances the interoceptive‐stimulus effects of cocaine, possibly due to facilitated increases in DA originating from NE terminals. Support: DA015040 and DA027535.
Differential Expression of MDPV‐Induced Taste Aversions and Thermoregulation in Adolescent and Adult Rats
Authors: Merluzzi Andrew Paul , Hurwitz Zachary , Briscione Maria , Cobuzzi Jennifer , Wetzell Bradley , Rice Kenner , Riley Anthony
Published: June 2021

As with all drugs of abuse, MDPV, a primary constituent in “bath salts,” is rewarding in rats. Yet little is known of its aversive effects, those which limit drug intake. Accordingly, the present experiment examined the ability of MDPV to induce taste aversions, an indicator of drug toxicity. Given the reported age differences in the sensitivity to the aversive effects of drugs, this assessment was made in adolescents and adults. Both groups were given access to a novel saccharin solution and then injected with various doses of MDPV. In addition, thermoregulatory changes were also observed following MDPV administration. Adolescent rats acquired significantly weaker taste aversions than adults and did so at a slower rate. Adolescent rats also showed a distinct hypothermic response compared to the adults. These results extend the findings with other drugs of abuse for which adolescents exhibit weaker aversions and different physiological responses following drug administration, possibly indicating an increased vulnerability. This research was supported in part by a grant from the Mellon Foundation, the Robyn Rafferty Scholarship and American University's Scholars and Artists Fellowship.
Stereoselective effects of methcathinone on intracranial self‐stimulation in rats
Authors: Bonano Julie Suyama , Banks Matthew L , Glennon Richard A , Negus S Stevens
Published: June 2021

Methcathinone (MCAT), the β‐keto analog of methamphetamine, is a potent central nervous system stimulant and parent compound to emerging drugs of abuse such as methylone, mephedrone, and MDPV (bath salts). Intracranial self‐stimulation (ICSS) is one type of procedure that has been used to assess abuse liability of stimulants and other classes of abused drugs. This study compared the potency, time course, and profile of effects produced by intraperitoneal administration of the (−) and (+) isomers of MCAT on ICSS in rats. Male Sprague Dawley rats were equipped with electrodes targeting the medial forebrain bundle, and responding during daily behavioral sessions was maintained by a range of 10 brain stimulation frequencies (56–158 Hz in 0.05 log steps). After saline treatment, brain stimulation maintained a frequency‐dependent increase in ICSS rates. Both (−)‐ and (+)‐MCAT produced a dose‐dependent and abuse‐related facilitation of ICSS that peaked after 10 min and was no longer evident after 300 min. (−)‐MCAT was approximately 3‐fold more potent than the less active (+) isomer. The stereoselectivity of MCAT effects on ICSS agrees with its stereoselectivity in producing other abuse‐related behavioral effects. These studies provide a foundation for future structure‐activity studies of abuse‐related effects produced by MCAT derivatives in this ICSS assay in rats. Funded by NIH grant R01 DA033930.
Unraveling mechanisms contributing to lack of antidepressant efficacy in juveniles and adolescents
Authors: Mitchell Nathan Clark , Horton Rebbeca E. , Vitela Melissa , Gould Georgianna G. , Koek Wounter , Daws Lynette C.
Published: June 2021

Depression is a major health problem for which most patients are not effectively treated. This problem is further compounded in children and adolescents where only two antidepressants [both selective serotonin reuptake inhibitors (SSRIs)] are currently approved for clinical use. Mouse models provide tools to identify mechanisms that might account for poor treatment response to antidepressants. However, until now the use of such models has been limited to studies in adult mice. Here we show that the tail suspension test can be used to assay antidepressant‐like effects of reference drugs in mice aged 21 (juvenile) and 28 (adolescent) days post‐partum, and that the antidepressant‐like activity of these drugs relates positively to expression levels of serotonin‐ and norepinephrine transporters. Importantly, we found that juvenile mice had markedly greater expression levels of plasma‐membrane monoamine transporter (PMAT) than adult mice. PMAT clears serotonin from extracellular fluid, and therefore may limit the therapeutic efficacy of SSRIs, providing a mechanistic basis for poor treatment response to SSRIs, particularly in juveniles and adolescents.
Anti‐neuroinflammaiton and antidepressant effects of Schisandrin B in mice
Authors: Yeh Ching‐Hua , Lin Ming‐Wei , Lin Hui‐Ching
Published: June 2021

Forced swimming exercise increases neuroinflammation and changes behavior thus taken as a murine depression‐like model. We investigated the effect of Schisandrin‐B (Schi‐B), one of the active components of Schisandra chinesnesis, on changing depressive behavior and neuroinflammation in mice forced to swim. Stress‐induced depression‐like model mice were prepared by a combination of chronic mild stress and forced swimming within one week. Depression‐like mice were gavaged with vehicle, Schi‐B (10 mg/kg) or Sertraline (32 mg/kg) twice a day for another one week. Sickness behavior, anhedonia, and the immobility time during 5 min of forced swimming test in mice were observed. In depression‐like mice, Schi‐B facilitated the recovery from anorexia, prevented anhedonia and decreased immobility time. Serum serotonin, mRNA expression of serotonin receptor and BDNF in Schi‐B‐treated mice were higher than those of vehicle‐treated mice. Schi‐B treatment decreased indoleamine 2, 3 dioxygenase (IDO) expression, microglia activation and inflammatory cytokines, TNF‐α, IL‐1β and IL‐6, in the brain of mice forced to swim. In conclusion, Schi‐B improved depression‐like behavior in mice forced to swim. The anti‐depressive effect of Schi‐B in mice is related to increase serotonin, serotonin receptor, and BDNF while decrease neuroinflammation.
Evaluating Food Front‐of‐Pack Labeling: a pan‐European Survey on Consumers’ Attitudes toward Food Labeling.
Authors: Gregori Dario , Ballali Simonetta , Gafare Claudia Elena , Stefanini Giulia , Halpern Georges M
Published: June 2021

Nutrition labels have been evaluated as mean to contribute to a reduction in the incidence and prevalence of diet‐related conditions. There is no convincing evidence that food labels are effective. “Computer Assisted Telephone Interview” (CATI) on population‐based sample of 16 European countries. Sociodemographic characteristics, knowledge on nutritional information and willingness to pay (WTP) were assessed. A total of 7550 closed interviews were analyzed. Advertising was the most efficient nutritional information tool by 28.7% of respondents, while labeling was favored by 10.6% of interviewees, regularly reading nutrition information in 71.8% of the cases. The majority of the respondent (70.2%) claimed to prefer as option for a FOP label nutritional information the “per portion” indication. WTP‐estimates provided an average accepted added price of 3.46 €. The majority of European consumers have a good general knowledge on nutritional information, and they have a well‐established understanding of health behaviours. Future mandatory labeling is perceived by European citizens as positive, although the perceived utility to pay for additional information was very low.
Effects of duloxetine in rats trained to discriminate between 22 and 2 hours food deprivation
Authors: Vanden Avond Mark A. , Johnson Amy R. , Reed Krystal A. , Savickas Monica M. , Nemec Dillon J. , Schultz Andrew T. , Hendrickson Elizabeth J. , Takle Nicole K. , Toddes Carlee A. , Lucas Ariel L. , Shattuck Britney N. , Halverson Andrew P. , Vossen Taylor J. , Jewett David C.
Published: June 2021

Duloxetine is a serotonin and norepinephrine reuptake inhibitor used clinically to treat depression and generalized anxiety disorder. We tested the effects of duloxetine in rats trained to discriminate 22 hr food deprivation from 2 hr food deprivation in a two‐lever, operant choice task. After rats acquired the discrimination, subjects were food restricted for 22 hr and administered saline and duloxetine (3.2–17.8 mg/kg, s.c.) before generalization tests. Food intake was measured for 1 hr after generalization tests. Duloxetine (10–17.8 mg/kg) significantly reduced the discriminative stimulus effects of 22 hr deprivation, rates of lever pressing, and food intake induced by 22 hr deprivation. The present findings are consistent with our previous research with sibutramine and offer further evidence that serotonin and norepinephrine are important regulators of both food intake and internal states associated with food deprivation.
Role of oxidative stress in anxiety‐like behavior and learning and memory impairment in a rat model of social stress
Authors: Patki Gaurav , Allam Farida , Bohat Ritu , Jafri Faizan , Saleem Mohammad , Salim Samina
Published: June 2021

Recently, we reported that direct pharmacological induction of oxidative stress causes anxiety‐like behavior and learning and memory impairment in a rodent model of oxidative stress (OS). In the present study, we tested whether induction of psychological stress using an established rat model of social stress leads to the same consequences i.e increased OS, anxiety and learning and memory impairment in rats. To test this hypothesis, a modified version of the resident‐intruder model for social stress (SS) was used. Sprague Dawley rats were randomly assigned to either SS or control exposure for 30 min for seven consecutive days. SS exposure resulted in intruder subordination, termed defeat, and was operationally defined by the intruder assuming a supine posture that was held for approximately 3 s. Controls were placed in a novel cage behind a partition for 30 min daily. At the end of the social defeat protocol, body weights, food and water intake were recorded, anxiety‐like behavior (open‐field and light‐dark) as well as learning‐memory (radial water maze) tests were conducted. We observed a significant decrease in the body weight in the SS rats as compared to the controls. Furthermore, social stress increased anxiety‐like behavior and caused learning and memory impairment of SS rats as compared to the control group. Biochemical data suggests involvement of oxidative stress mechanisms in these behaviors. NIH R15G103327
Transcriptional regulation of human UDP‐glucuronosyltransferase 2B4 (UGT2B4) by sulfotransferase deficiency
Authors: Barrett Kathleen , Fang Hailin , Kocarek Thomas A , Runge‐Morris Melissa
Published: June 2021

We previously found that UGT2B4 expression is up‐regulated in HepG2 cells with reduced sulfotransferase activity as achieved by stable knockdown of 3′‐phosphoadenosine 5′‐phosphosulfate synthases 1 and 2 (PAPSS1/2). Since UGT2B4 is reportedly regulated by the bile acid‐sensing nuclear receptor FXR, we investigated whether PAPSS1/2 knockdown‐inducible UGT2B4 expression is mediated through FXR. When a luciferase reporter plasmid containing 2Kb of the UGT2B4–5′‐flanking sequence was transiently transfected into PAPSS1/2 knockdown HepG2 (shPAPSS) cells, luciferase activity was significantly (~4‐fold) higher than it was after transfection into control cells. Treatment of shPAPSS cells with an FXR agonist (GW4064 or chenodeoxycholic acid) or transfection with an FXR expression plasmid increased reporter activity (~2‐to 5‐fold), demonstrating the FXR responsiveness of 2Kb‐UGT2B4‐Luc. However, knockdown of FXR in shPAPSS cells had no effect on 2Kb‐UGT2B4‐Luc activity, and mutation of a previously reported FXR response element at nt ‐1180 reduced reporter activity by only ~20%. These results indicate that the information conferring transcriptional up‐regulation of UGT2B4 in response to PAPSS1/2 knockdown is contained within the first 2Kb of the UGT2B4 5′‐flanking region but that FXR is not responsible for this transcriptional activation. This work was supported by NIH grant ES005823.
Functional association of hepatocyte nuclear factor 4 alpha (HNF4α) and Krüppel‐like factor 9 (KLF9) in cytochrome P450 2D6 (CYP2D6) regulation during pregnancy
Authors: Koh Kwi Hye , Pan Xian , Yu Aiming , Jeong Hyunyoung
Published: June 2021

CYP2D6 is responsible for metabolism of ~25% of marketed drugs. Clinical data indicate that pregnancy increases CYP2D6‐mediated drug metabolism. Previously, we have found humanized CYP2D6 transgenic mice (tg‐CYP2D6) as a potential model to study CYP2D6 induction during pregnancy. To elucidate the mechanisms underlying CYP2D6 regulation during pregnancy, we obtained a list of genes differentially expressed in livers of pregnant vs. nonpregnant tg‐CYP2D6 mice through microarray experiments. The results revealed that 430 and 225 genes were up‐or down‐regulated, respectively, during pregnancy. The upregulated genes included the following 11 transcription factors: Ar, Ascl1, Atf5, Ddit, Egr1, Foxa3, Junb, Klf9, Klf10, Mafk and Rev‐erbα. Most of these transcription factors, when individually transfected into HepG2 cells, did not significantly increase promoter activities of CYP2D6. However, when co‐transfected with a known transcriptional activator of CYP2D6 (i.e., HNF4α), KLF9 significantly increased HNF4α transactivation of CYP2D6; HNF4α alone enhanced CYP2D6 promoter activity by 1.7‐fold, whereas co‐transfection of HNF4α and KLF9 enhanced the activity by 12.6‐fold. Together, our results suggest that the increased expression of KLF9 may be in part responsible for CYP2D6 induction during pregnancy by potentiating HNF4α transactivation. This study is supported by NIH (R01HD065532).
Functional analysis of splice variants of human constitutive androstane receptor: Investigation with flavonol (3‐hydroxyflavone) and its metabolites
Authors: Lau Aik Jiang , Chang Thomas K. H.
Published: June 2021

Constitutive androstane receptor (CAR) controls the transcription of genes involved in diverse biological functions, including bioactivation and detoxification of drugs and toxicants. Naturally occurring splice variants of human CAR (hCAR) exist, including hCAR‐SV23 (insertion of amino acids SPTV), hCAR‐SV24 (APYLT), and hCAR‐SV25 (SPTV and APYLT). In the present study, we investigated the effect of flavonol (3‐hydroxyflavone) and its metabolites (galangin, datiscetin, kaempferol, morin, quercetin, isorhamnetin, tamarixetin, myricetin, and syringetin) on the functionality of hCAR‐SV23, hCAR‐SV24, and hCAR‐SV25. As assessed in a cell‐based reporter gene assay, only flavonol activated hCAR‐SV23 and hCAR‐SV24, whereas none of them activated hCAR‐SV25. Flavonol did not recruit steroid receptor coactivators SRC‐1, SRC‐2, or SRC‐3 to the ligand‐binding domain of hCAR‐SV23 or hCAR‐SV24. By comparison, flavonol, galangin, datiscetin, kaempferol, quercetin, isorhamnetin, and tamarixetin activated hCAR‐WT, but only flavonol recruited coactivators. Thus, addition of OH or OCH3 substituent at the C2′ or C5′ position of flavonol abolished activation of hCAR‐WT. In summary, flavonol and its metabolites differentially activated hCAR isoforms and, in some cases, this occurred by a mechanism that did not involve coactivator recruitment. [Supported by CIHR and MSFHR]
P‐glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2) restrict the brain penetration of the Janus family of tyrosine kinase (JAK) inhibitor CYT387
Authors: Durmus Selvi , Xu Ning , Sparidans Rolf W , Wagenaar Els , Beijnen Jos H , Schinkel Alfred H
Published: June 2021

CYT387 is an oral Janus family of tyrosine kinase (JAK) 1/2 inhibitor and currently undergoes Phase I/II clinical trials for myelofibrosis and myeloproliferative neoplasm treatment. We aimed to establish whether the multidrug efflux transporters P‐glycoprotein (ABCB1) and Breast Cancer Resistance Protein (ABCG2) restrict oral availability and brain penetration of CYT387. To investigate this, plasma exposure and brain concentrations of CYT387 were analyzed after oral administration of CYT387 to wild type (WT), Abcb1a/1b−/−, Abcg2−/− and Abcb1a/1b−/−;Abcg2−/− mice. Over 8 hours, plasma exposure of CYT387 was similar between all the strains, indicating that these transporters do not limit oral availability of CYT387. Despite the similar systemic exposure, brain concentrations of CYT387 were increased ~10‐fold in the Abcb1a/1b−/−;Abcg2−/− mice, but not in the Abcb1a/1b−/− or Abcg2−/− mice, compared to the WT strain. However, in Abcg2−/− mice brain levels of CYT387 were slightly, albeit not significantly increased, suggesting that CYT387 might be a better substrate of Abcg2 rather than Abcb1a at the blood‐brain barrier. These results overall indicate a marked and additive role of Abcg2−/− and Abcb1a/1b−/− in restricting brain penetration of CYT387.
Assessing drug interaction risk of the grapefruit juice component and dietary supplement 6′,7′‐dihydroxybergamottin via physiologically‐based pharmacokinetic modeling and simulation
Authors: Ainslie Garrett Robert , Gufford Brandon T , Won Christina S , Wolf Kristina K , Paine Mary F
Published: June 2021

The grapefruit juice (GFJ) component 6′7′‐dihydroxybergamottin (DHB) is an irreversible intestinal CYP3A inhibitor, present in amounts up to 4.7 mg/240 mL GFJ. ‘Pharmacokinetic boosting’ effects of GFJ on drug exposure fueled marketing of DHB as a dietary supplement. DHB product labeling ranges from 50–300 mg, raising concerns for amplified drug interactions. Interaction risk with the CYP3A substrate and μ‐opioid receptor agonist loperamide was assessed via physiologically‐based pharmacokinetic modeling and simulation using Simcyp. Inhibition kinetics of DHB toward loperamide N‐demethylation were recovered using human intestinal microsomes. Partitioning, binding, and permeability parameters for loperamide and DHB were predicted from physicochemical properties. Results were compared to plasma concentration‐time data from an in‐house clinical GFJ‐loperamide interaction study (3.6 mg DHB/240 mL GFJ; 16 mg loperamide). Effects of higher DHB doses were simulated. Median predicted loperamide AUC (93 nM*h), Cmax (5.2 nM), and tmax (2.6 h) agreed with observed values (101 nM*h, 6.0 nM, and 3.0 h, respectively). Simulations with 50 and 300 mg DHB predicted a 2.2‐and 3.3‐fold median increase in loperamide AUC, respectively. Modeling and simulation informed design of a clinical DHB supplement‐loperamide interaction study and provides a framework for DHB supplement‐drug interaction risk assessment.
Curcumin and turmeric as preventive agents for iron‐related carcinogenesis.
Authors: Messner Donald J , Robinson Todd , Kowdley Kris V.
Published: June 2021

Iron overload is a risk factor for liver cancer. The aim of this study was to determine whether curcumin (diferuloylmethane) and related compounds, previously shown to bind iron and reduce iron toxicity in cultured cells, can also prevent iron‐related carcinogenesis in vitro. Curcumin is derived from the food spice turmeric; our test agents included a defined mixture of purified curcuminoids and a standardized turmeric (C. longa) extract. Both preparations reduced oxidative stress (TBARS assay) and cytotoxicity (mtt assay) associated with iron overload in T51B cells, a non‐neoplastic rat liver cell line. Iron (ferric ammonium citrate, FAC) acted as a tumor promoter in this model: neoplastic transformation (as judged by growth in soft agar) was increased by initiation with 0.5 μg/ml MNNG followed by 16 week culture with 200 μM FAC (p<0.05). This was reduced (p<0.05) by coadministration (with FAC) of 20 μM purified curcuminoids or the turmeric extract. Effects at lower concentrations were less consistent. Curcuminoid levels in the cells peaked in the first 4 hours of treatment and declined with a half‐life of 1–2 days. Depending partly on dosing frequency, inhibition of cell transformation could be maintained through 20 weeks of iron exposure. These results demonstrate that curcumin can prevent iron‐related carcinogenesis in vitro. They justify and inform further study in vivo. Supported by NIH grant AT03448.
Purinergic Mechanisms in Breast Cancer Development: A Role for Nucleoside Diphosphate Kinase
Authors: Wong Senny , Yokdang Nucharee , Speirs Katie , Buxton Iain L.O.
Published: June 2021

A secreted factor from breast cancer cells, nucleoside diphosphate kinase (NDPK) was shown to promote cell migration, proliferation, and tumor‐mediated angiogenesis, which may support metastasis. The objective of this study was to observe breast cancer development in mice in the presence of inhibitors of purinergic signaling. A luciferase‐tagged murine breast adenocarcinoma 4T1 cell line was injected into the mammary fat of immune competent BALB/C mice that were divided into treatment groups receiving a combination of ellagic acid (EA; NDPK inhibitor) and/or MRS2179 (P2Y1 receptor antagonist) or endostatin (angiogenesis inhibitor as positive control). Tumor growth was measured by imaging tumor luminescence using a Lumina II (Caliper Life Sci.). Serum samples were collected for detection of NDPK levels by enzyme‐linked immunosorbent assay. We found a significant decrease in the primary tumor growth rate at days 21 and 28 with all treatments versus the control. Future work will focus on NDPK present in the serum of women with breast cancer to support or reject the hypothesis that secreted NDPK has a role during early primary tumor and metastasis development and could serve as a biomarker of disease. (Supported by NIH HD053028)
Bioenergetic immunostimulation, metabolism, and nutrition support in lung cancer: a case study
Authors: TULP ORIEN LEE , Einstein George P , Kowaleski Janusz , Dancewicz Maciej
Published: June 2021

The most common type of cancer [Ca]in the USA is lung Ca, where it contributes to >; 150,000 deaths annually despite many advances in early diagnosis and treatment modalities. The patient was a 64 y.o. WM, no prior Hx of Ca or other major illness. Patient underwent lobectomy of a 5.4 cm mass R lung which confirmed Ca, followed by a vigorous bioenergetics regimen including Nutritional [macrobiotic: 65% mixed grains, 20% mixed veg, 15% fruits, exercise [<800 m swimming daily], and maintaining a positive attitude. Pt resumed full time work after 2 w, and remains Ca‐free and in excellent health at 7 y followup.

Conclusion

Patients require adequate nutritional, physical and emotional Rx following lobectomy for lung Ca. Bioenergic Rx may result in improved CD4, CD25, & T cell function and decrease mortality & improve survival in Lung Ca. Institutional Support.
Crude extracts of Antrodia cinnamomea inhibit invasion and migration of Human Hepatocellular Carcinoma Cells
Authors: Chen Ying‐Yi , Sheu Ming‐Jyh
Published: June 2021

A native fungus native to Taiwan, Antrodia cinnamomea is believed to be effective in preventing many diseases including apoptosis of different kinds of cancer cells like NSCLC lung cancer, breast cancer, and leukemia cancer. In this study we used the ethanol extract of Antrodia cinnamomea (EEAC) to evaluate its effects on anti‐proliferative and anti‐metastatic activity of human hepatocellular liver cancer cells HepG2 and Hep3B cells. The underlying mechanism was also conducted by Western blotting analysis. The results of MTT assay revealed non‐cytotoxic concentrations Antrodia cinnamomea dose and time‐dependently inhibited the migration of HepG2 and Hep3B cells. By gelatin zymography assay, EEAC effectively inhibited MMP‐9 and MMP‐2 activities. By Western blotting analysis, treatment with EEAC decreased phosphatidylinositol‐3‐kinase (PI3K)/AKT, MMP‐2 and MMP‐9 expressions in HepG2 and Hep3B cells. EEAC also exerted an inhibitory effect on the phosphorylation of extracellular signal‐regulated kinases 1 and 2 (ERK1/2) and inhibition of activation of nuclear factor kappa B (NF‐κB), c‐Fos, and c‐Jun. According to the above data, the growth inhibition of human hepatocellular liver cancer cells by EEAC was mediated by induction of migration and cell proliferation related proteins. In conclude, Antrodia cinnamomea may be a potential candidate for treating those patients with liver cancer metastasis.
Secreted factors of Lactobacillus rhamnosus GG culture prevents chronic alcohol‐induced liver injury
Authors: Wang Yuhua , Wang Cuiling , Wang Chunhong , Zhang Min , Liu Yanlong , McClain Craig , Feng Wenke
Published: June 2021

Lactobacillus rhamnosus GG (LGG) culture supernatant (LGGs) ameliorated acute alcohol‐induced intestinal permeability and liver injury. We examined whether LGGs provides a protective effect. Mice were fed diet containing 5% alcohol for 4 weeks, and LGGs was administered at a dose of equivalent to 109 CFU/mouse with alcohol. LGGs decreased chronic alcohol‐induced plasma LPS concentration, increased expression of mucus factors and tight junctions, and decreased intestinal permeability. LGGs decreased chronic alcohol‐induced liver injury evaluated by ALT in plasma. Alcohol‐induced hepatic steatosis was prevented by LGGs evaluated by triglyceride contents and Oil Red O staining. Alcohol decreased AMPK and ACC phosphorylation and CPT‐1 expression, and these effects were prevented by LGGs administration in vivo and in cell culture. Inhibition of AMPK phosphorylation by Compound C abolished LGGs‐induced ACC inactivation and CPT‐1 up‐regulation, suggesting that the protective effect of LGGs on lipid metabolism in response to alcohol exposure is mediated via AMPK pathway. LGGs prevented alcohol‐induced hepatic apoptosis evaluated by TUNEL assay. These results suggest that LGGs is effective in preventing chronic alcohol‐induced liver injury by promoting intestinal integrity and attenuating hepatic apoptosis, and increasing lipid β‐oxidation through AMPK phosphorylation.
S‐Adenosyl‐L‐methionine (SAMe) Protects the Liver from Acetaminophen (APAP) Protein modifications due to Oxidative Stress
Authors: Lamyaithong Andre Benja , Brown J. Mike , Ball John G , Wolfe Jacob , Valentovic Monica A
Published: June 2021

Acetaminophen (APAP) overdose induces hepatotoxicity due to biotransformation to a toxic metabolite which increases oxidative stress. This study tested whether APAP causes 3‐nitrotyrosine (3‐NT) formation in mitochondria and cytosol that would be modified by S‐Adenosyl‐L‐methionine (SAMe). Male mice were randomly divided into 4 groups (N=5–10/group) and injected intraperitoneal (ip) as indicated: vehicle (VEH, 15 ml/kg water ip), SAMe (1.25 mmol/kg 5 ml/kg ip), APAP (250 mg/kg ip), SAMe and APAP (SAMe administered 1h after APAP). Livers were collected 4 h following APAP. Subcellular fractions were isolated from mitochondria and cytosol for Western analysis of Mn and Cu/Zn SOD protein expression in mitochondria and cytosolic fractions. Additional studies evaluated 3‐NT adduction of mitochondrial and cytosolic proteins. SAMe administered 1 h after APAP reduced APAP hepatic toxicity when measured 4 h post APAP injection. Mn SOD and Cu/Zn SOD levels were detected in mitochondrial and cytosolic fractions. 3‐NT adduction was increased by APAP in mitochondrial fractions. SAMe reduced APAP associated increase in 3‐NT adduction of mitochondrial proteins. In conclusion, APAP induced nitrosative stress is reduced by SAMe especially in the mitochondria and mitochondrial MnSOD protein expression was not depressed by APAP treatment. (Supported by NIH Grants 3P20RR016477–09S4, 5P20RR016477 and 8P20GM103434).
Potentiation of cytotoxicity by two metabolites of 3‐(3,5‐dichlorophenyl)‐2,4‐thiazolidinedione (DCPT) in HepG2 cells
Authors: Ejaz Sadaff , Tchao Ruy , Harvison Peter J
Published: June 2021

Previously, we showed that 3‐(3,5‐dichlorophenyl)‐2,4‐thiazolidinedione (DCPT) is cytotoxic in human hepatoma HepG2 cell line. DCPT is converted into [[(3,5‐dichlorophenyl)amino]carbonyl]thioglycolic acid (DCTA), which can potentially degrade into 3,5‐dichlorophenyl isocyanate (DPI) and mercaptoacetic acid (MAA). The goal of these studies was to determine which metabolite of DCPT is the proximal agent for toxicity. HepG2 cells were therefore incubated with 200 μM DCPT, DCTA, DPI, MAA or a combination of DPI plus MAA. Cell viability and proliferation were assessed over a 24 hr period. DCTA and the combination treatment (DPI plus MAA) were significantly more cytotoxic at 24 hr (~65% cell loss) than DCPT, DPI or MAA alone. To further study the cytotoxicity of the combination treatment (DPI plus MAA), HepG2 cells were pretreated (4 hr) with either 200 μM DPI or MAA, and then challenged with the alternative compound. The DPI pretreatment followed by MAA caused a loss in cell viability beginning at 2 hr. On the other hand, pretreatment with MAA followed by DPI caused a loss in viability at 24 hr. Our results suggest that the presence of both hydrolytic products of DCTA may contribute to DCPT cytotoxicity in HepG2 cells. Furthermore, there appears to be an additive/synergistic cytotoxic effect between DPI and MAA. Supported by PHS grant ES012499 and the USciences Department of Pharmaceutical Sciences.
Effects of popcorn butter flavorings, diacetyl and 2,3‐pentanedione, on isolated airway preparations
Authors: Zaccone Eric J , Thompson Janet A , Shimko Michael J , Fedan Jeffrey S
Published: June 2021

RATIONALE

“Popcorn workers’ lung” is a life‐threatening, fixed obstructive pulmonary disease caused by inhalation of butter flavoring vapor. Histological evidence from animals exposed by inhalation to the butter flavorings, diacetyl (2,3‐butanedione), and a diacetyl substitute, 2,3‐pentanedione (acetyl proprionyl), demonstrated vapor‐concentration‐dependent damage to epithelial cells. Because nothing is known about the effects of diacetyl and 2,3‐pentanedione on airway smooth muscle (ASM), we investigated the pharmacological activity of these two flavorings on isolated airways.

METHODS

In rat isolated, perfused tracheas, the contractile and relaxant effects of diacetyl and 2,3‐pentanedione applied to the serosal and mucosal surfaces of intact and denuded contracted (MCh; 3×10−5 M) tracheas were evaluated.

RESULTS

Concentrations of both agents greater than 3 mM resulted in ASM relaxation. Relaxation of the trachea by diacetyl and 2,3‐pentanedione was not dependent upon the presence of the epithelium.

CONCLUSIONS

Our findings indicate that both flavorings are similar in their effects as ASM relaxants.
The role of the ß2‐adrenoceptor Gs pathway in the development of the asthma phenotype in murine models
Authors: FORKUO GLORIA S , Thanawala Vaidehi J , Sawalha Nour , Knoll Brian , Bond Richard
Published: June 2021

Asthma is a chronic inflammatory disease of the airways. We have shown the requirement of the β2AR in the development of three main features of asthma in murine models. β2AR signal through various pathways including the canonical Gs and the β‐arrestin pathway. The objective of this study was to determine if formoterol and salmeterol, documented to have different relative intrinsic efficacies at the Gs pathway resulted in different asthma phenotypes. We used wild type (WT) and phenylethanolamine‐N‐methyltransferase null mice (PNMT−/−) mice. PNMT is responsible for converting norepinephrine into epinephrine and null mice have no detectable levels of epinephrine, the endogenous ligand for ß2AR. PNMT−/− mice were treated with formoterol and salmeterol 10μg/kg/day and 3μg/kg/day respectively, intraperitoneally twice daily at four treatment durations. Mice were phenotyped in an ovalbumin sensitized and challenged (Ova S/C) model. WT, but not PNMT−/− mice, had significant increases in inflammatory cells, mucus hyper‐secretion and airway hyper‐responsiveness. Ova S/C PNMT−/− mice, treated with formoterol and salmeterol significantly restored the asthma phenotype to the level seen in WT mice. Thus drugs with differing efficacies at the Gs pathway are equal at restoring the asthma phenotype, and suggest this pathway is not responsible for the requirement of ß2AR activation in development of the asthma phenotype.