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The effects of growth hormone on opioid-induced toxicity in vitro
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There is an ongoing opioid crisis in the United States that is portrayed by a large number of opioid-related deaths. Many of these cases involve commonly used prescription opioids, such as morphine, oxycodone, fentanyl, and methadone. This is concerning and highlights the problems associated with long-term opioid treatment. In addition to opioid-related deaths, long-term opioid use may impact higher brain functions, such as cognitive function. The cause of cognitive decline following opioid treatment may be associated with increased neuronal cell death, inhibited neurogenesis, and altered volumes of specific brain regions important for cognition. Growth hormone (GH), a pituitary hormone regulated by the hypothalamic somatotropic axis, may counteract several of these effects. The hormone, alongside with its mediator insulin-like growth factor-1 (IGF-1), is associated with pro-cognitive effects and display promising neuroprotective actions in the CNS. The main aim for this thesis was to examine the impact of opioids on cell viability and the potentially protective, restorative, and effects linked to pro-cognitive properties of GH in mixed neuronal cell cultures and cell lines. The results clearly display that specific opioids, such as methadone, decrease cell viability, possibly via negative effects on mitochondrial morphology. GH treatment alleviated the negative effects of methadone in cortical cell cultures as well as successfully restored mitochondrial and membrane integrity past injury. Moreover, GH treatment to primary hippocampal cell cultures increased the number of dendritic spines, which are linked to higher cognitive functions, indicating that the hormone act as a cognitive enhancer in the CNS. In conclusion, this thesis provides further evidence that opioids negatively impact cell viability, an effect that may underlie reduced cognitive function as seen in several patients consuming opioids-long term. GH was able to counteract these effects and also able to restore damaged cellular functions. This thesis further confirms the essential role of GH in acting as a cognitive enhancer in the CNS, highlighting the potential role of GH as a treatment for cognitive dysfunctions.    

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 60
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 279
Keywords [en]
Growth hormone, opioids, methadone, morphine, ketobemidone, fentanyl, oxycodone, hydromorphone, insulin-like growth factor, cell viability, NG108-15, SH-SY5Y, hippocampus, cortex
National Category
Cell and Molecular Biology Pharmaceutical Sciences Pharmacology and Toxicology
Research subject
Pharmaceutical Science
Identifiers
URN: urn:nbn:se:uu:diva-393940ISBN: 978-91-513-0765-7 (print)OAI: oai:DiVA.org:uu-393940DiVA, id: diva2:1357898
Public defence
2019-11-22, B21, Biomedicinskt centrum (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-10-30 Created: 2019-10-04 Last updated: 2019-11-12
List of papers
1. Mitochondrial function and membrane integrity: an in vitro comparison between six commonly used opioids
Open this publication in new window or tab >>Mitochondrial function and membrane integrity: an in vitro comparison between six commonly used opioids
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Introduction: There is an ongoing opioid crisis in the United States where the illicit and non-medical use of prescription opioids is associated with an increasing number of overdose deaths. Few studies have investigated the effects of opioid-induced effects on cell viability, and comparative studies are scarce. Here we examine the toxic effect on cell viability from six commonly used opioids; methadone, morphine, oxycodone, hydromorphone, ketobemidone, and fentanyl with respect to mitochondrial and membrane function in vitro. Methods: The opioids were tested in four different cell cultures; primary cortical cell cultures, human neuroblastoma SH-SY5Y cells, and both differentiated and undifferentiated neuroblastoma/glioma hybrid NG108-15 cells. Results: The six different opioids displayed the same trend of reduced cell viability in all four cell cultures. The ranking of opioids, with respect to reduced cell viability were as follows; methadone, fentanyl, ketobemidone, oxycodone, hydromorphone, and morphine. Conclusion: Methadone was ranked as the most toxic opioid closely followed by fentanyl. Ketobemidone and oxycodone had modest effects while both hydromorphone and morphine only displayed little to no negative impact on cell viability.

Keywords
Methadone, Morphine, Oxycodone, Hydromorphone, Ketobemidone, Fentanyl, Cell viability, primary cell cultures, NG108-15, SH-SY5Y, Opioids
National Category
Basic Medicine
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-393205 (URN)
Available from: 2019-09-17 Created: 2019-09-17 Last updated: 2019-10-04
2. The effects of morphine, methadone, and fentanyl on mitochondrial morphology: a live cell imaging study
Open this publication in new window or tab >>The effects of morphine, methadone, and fentanyl on mitochondrial morphology: a live cell imaging study
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The important role of mitochondria in maintaining normal brain cell function has been demonstrated in several neurodegenerative diseases where mitochondrial dysfunction is a prominent feature. Accumulating evidence indicates that opioids may induce neuronal cell death and inhibit neurogenesis, two factors that are dependent on normal mitochondrial function. The aim of the present study was to examine the effects of morphine, methadone, and fentanyl on mitochondrial morphology. Cells from the neuroblastoma/glioma hybrid cell-line NG108-15 were seeded on 96-well cell culture plates and treated with MitoTracker™ for 30 min prior to opioid treatment. Morphine, methadone, and fentanyl were added at various concentrations and images of mitochondria were acquired every 30 min for four hours using a high-content imaging device. The morphological parameters total mitochondrial area, mitochondrial network, number of mitochondrial objects, and the mean area of mitochondrial objects were analyzed using automated image analysis. Methadone and fentanyl, but not morphine, decreased the mitochondrial network, the number of mitochondrial objects, and increased the mean area of mitochondrial objects. Both methadone and fentanyl altered mitochondrial morphology with no effects seen from morphine treatment. These data suggest that methadone and fentanyl disrupt mitochondrial morphology, which may contribute to neuronal cell death.

Keywords
Cell culture, Fentanyl, Live cell imaging, Methadone, Mitochondria, Morphine, Morphology, NG108-15, Opioids, Time-lapse
National Category
Cell and Molecular Biology
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-393936 (URN)
Funder
Swedish Research Council, 9459The Swedish Brain Foundation
Available from: 2019-09-30 Created: 2019-09-30 Last updated: 2019-10-04
3. Growth hormone is protective against acute methadone-induced toxicity by modulating the NMDA receptor complex
Open this publication in new window or tab >>Growth hormone is protective against acute methadone-induced toxicity by modulating the NMDA receptor complex
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2016 (English)In: Neuroscience, ISSN 0306-4522, E-ISSN 1873-7544, Vol. 339, p. 538-547Article in journal (Refereed) Published
Abstract [en]

Human growth hormone (GH) displays promising protective effects in the central nervous system after damage caused by various insults. Current evidence suggests that these effects may involve N-methyl-d-aspartate (NMDA) receptor function, a receptor that also is believed to play a role in opioid-induced neurotoxicity. The aims of the present study were to examine the acute toxic effects of methadone, an opioid receptor agonist and NMDA receptor antagonist, as well as to evaluate the protective properties of recombinant human GH (rhGH) on methadone-induced toxicity. Primary cortical cell cultures from embryonic day 17 rats were grown for 7 days in vitro. Cells were treated with methadone for 24 h and the 50% lethal dose was calculated and later used for protection studies with rhGH. Cellular toxicity was determined by measuring mitochondrial activity, lactate dehydrogenase release, and caspase activation. Furthermore, the mRNA expression levels of NMDA receptor subunits were investigated following methadone and rhGH treatment using quantitative PCR (qPCR) analysis. A significant protective effect was observed with rhGH treatment on methadone-induced mitochondrial dysfunction and in methadone-induced LDH release. Furthermore, methadone significantly increased caspase-3 and -7 activation but rhGH was unable to inhibit this effect. The mRNA expression of the NMDA receptor subunit GluN1, GluN2a, and GluN2b increased following methadone treatment, as assessed by qPCR, and rhGH treatment effectively normalized this expression to control levels. We have demonstrated that rhGH can rescue cells from methadone-induced toxicity by maintaining mitochondrial function, cellular integrity, and NMDA receptor complex expression.

Place, publisher, year, edition, pages
Elsevier, 2016
Keywords
growth hormone, methadone, opioids, neuroprotection, NMDA, primary cell culture
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-306727 (URN)10.1016/j.neuroscience.2016.10.019 (DOI)000389168500045 ()27746341 (PubMedID)
Funder
Swedish Research Council, 9459The Swedish Brain Foundation
Available from: 2016-11-03 Created: 2016-11-03 Last updated: 2019-10-04
4. The Protective and Restorative Effects of Growth Hormone and Insulin-Like Growth Factor-1 on Methadone-Induced Toxicity In Vitro
Open this publication in new window or tab >>The Protective and Restorative Effects of Growth Hormone and Insulin-Like Growth Factor-1 on Methadone-Induced Toxicity In Vitro
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2018 (English)In: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 19, no 11, p. 1-16, article id ijms-387278Article in journal (Refereed) Published
Abstract [en]

Evidence to date suggests that opioids such as methadone may be associated with cognitive impairment. Growth hormone (GH) and insulin-like growth factor-1 (IGF-1) are suggested to be neuroprotective and procognitive in the brain and may therefore counteract these effects. This study aims to explore the protective and restorative effects of GH and IGF-1 in methadone-treated cell cultures. Primary cortical cell cultures were harvested from rat fetuses and grown for seven days in vitro. To examine the protective effects, methadone was co-treated with or without GH or IGF-1 for three consecutive days. To examine the restorative effects, methadone was added for the first 24 h, washed, and later treated with GH or IGF-1 for 48 h. At the end of each experiment, mitochondrial function and membrane integrity were evaluated. The results revealed that GH had protective effects in the membrane integrity assay and that both GH and IGF-1 effectively recovered mitochondrial function and membrane integrity in cells pretreated with methadone. The overall conclusion of the present study is that GH, but not IGF-1, protects primary cortical cells against methadone-induced toxicity, and that both GH and IGF-1 have a restorative effect on cells pretreated with methadone.

Keywords
growth hormone; insulin-like growth factor-1; neuroprotection; neurorecovery; cognition; primary cell cultures; methadone; opioids
National Category
Basic Medicine Cell and Molecular Biology
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-369995 (URN)10.3390/ijms19113627 (DOI)000451528500343 ()30453639 (PubMedID)
Funder
Swedish Research Council, 9459The Swedish Brain Foundation
Note

Även finansierat av Kjell och Märta Beijer stiftelsen.

Available from: 2018-12-18 Created: 2018-12-18 Last updated: 2019-10-04Bibliographically approved
5. Growth hormone increases dendritic spine density in primary hippocampal cell cultures
Open this publication in new window or tab >>Growth hormone increases dendritic spine density in primary hippocampal cell cultures
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Objective: Growth hormone (GH) is widely known for its peripheral effects during growth and development. However, numerous reports also suggest that GH exert pro-cognitive, restorative, and protective properties in the brain. In in vitro studies, the detection of dendritic spines, small protrusions extending from axons, can act as a marker for cognition-related function as spine formation is considered to be associated with learning and memory. Here we show that an acute 24-hour treatment of GH can increase dendritic spine density in primary hippocampal cell cultures.

Design: Primary hippocampal cells were harvested from embryonic Wistar rats and cultured for 14 days. Cells were treated with supra-physiological doses of GH (10-1000 nM) and subjected to a high-throughput screening protocol. Images were acquired and analyzed using automated image analysis and the number of spines, spines per neurite length, neurite length, and mean area of spines, was reported.

Results: GH treatment increased dendritic spine density using the highest dose while the general health of the cells was unaffected.

Conclusion: The results from the present study further confirms a potential role of GH in the treatment of cognitive dysfunction.

Keywords
growth hormone, dendritic spines, spine formation, cognition, memory, high-throughput screening, hippocampus, cell culture, in vitro
National Category
Cell and Molecular Biology
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-393938 (URN)
Funder
The Swedish Brain FoundationSwedish Research Council, 9459
Available from: 2019-09-30 Created: 2019-09-30 Last updated: 2019-10-04

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