Induction of CYP3A4 mRNA in the cell lines HepG2 and Colo320 by antiretroviral drugs

Das primäre Ziel dieser Arbeit ist es, In-Vitro nachzuweisen, dass antiretrovirale Wirkstoffe ver-schiedener Substanzklassen die Bildung der CYP3A4-mRNA in den Zelllinien HepG2 und Co-lo320 induzieren können. Zusätzlich wurde die CYP3A4-Expression in verschiedenen Zellkultu-ren anhand der mRNA- Bestimmung untersucht. Für die Versuchsreihe wurden die Zelllinien HeLa, HepG2, Caco-2, Colo320 und humanes Le-bergewebe auf ihren Gehalt an CYP3A4-mRNA getestet. Die Inkubationsversuche wurden mit den Zelllinien HepG2 und Colo320 durchgeführt. Getestet wurden die antiretroviralen Substan-zen Abacavir, Amprenavir, Zidovudin Didanosin, Stavudin, Saquinavir, Zalcitabin, Efavirenz, Nevirapin, Delavirdin, Indinavir, Lopinavir/Ritonavir und Nelfinavir. Als Referenzsubstanz für die Wirkung auf die CYP3A4-mRNA-Bildung wurde der bekannte CYP3A4-Induktor Rifampi-cin eingesetzt. Die Inkubationsversuche erfolgten auf Zellkulturplatten mit 6 Kavitäten. Jede Ka- vität enthielt etwa eine Million Zellen. Die Zellen wurden 20 Stunden bei 37°C mit der Testsub-stanz in unterschiedlichen Konzentrationen inkubiert. Nach erfolgter Inkubation wurde die ge-samte RNA aus den Zellen in einem isoliert und in cDNA konvertiert. Die Quantifizierung der CYP3A4-mRNA erfolgte mittels Real-Time-PCR am LightCycler (Roche) über einen externen Standard, der in der Sequenz mit der gemessenen CYP3A4-mRNA identisch war. Die untersuchten nukleosidischen Reverse-Transkriptase-Inhibitoren (Abacavir, Didanosin, Lamivudin, Stavudin, Zalcitabin und Zidovudin) induzierten die Bildung von CYP3A4-mRNA nicht. In der Gruppe der nicht-nukleosidischen Reverse- Transkriptase-Inhibitoren konnte für die Substanzen Efavirenz und Delavirdin eine signifikante Induktion der CYP3A4-Expression nach-gewiesen werden, nicht jedoch für Nevirapin. Für die untersuchten Proteaseinhibitoren Amprenavir, Indinavir, Nelfinavir, Ritonavir und Saquinavir konnte eine Induktion der CYP3A4-Expression in den Zelllinien HepG2 und Colo320 gezeigt werden. Die Substanzkom-bination des Proteaseinhibitors Kaletra® (Lopinavir/Ritonavir) führte in HepG2-Zellen nicht, jedoch in Colo320-Zellen, zur Induktion der CYP3A4-Genexpression. In den hier dargestellten Versuchen konnte gezeigt werden, dass der CYP3A4-mRNA-Gehalt in Zellen der Zelllinie HepG2 mit zunehmender Passagezahl abnimmt. Auch konnte nachgewiesen werden, dass die Zelllinien HepG2 und Colo320 geringere Mengen an CYP3A4-mRNA als Zellen humanen Le-bergewebes enthalten. Die in dieser Arbeit dargestellten Inkubationsversuche wurden erstmalig mit den Zelllinien HepG2 und Colo320 durchgeführt. Mit dieser Arbeit gelang es zum Zeitpunkt der Experimente eine induktive Potenz für die untersuchten Proteaseinhibitoren nachzuweisen. So zeigen die Er-gebnisse der hier vorgestellten Arbeit trotz der bekannten inhibitorischen Wirkung der Proteas-einhibitoren eine Zunahme des CYP3A4-mRNA in den Zellen und somit ein wenn auch schwach ausgeprägtes induktives Potential der Wirkstoffe. Dabei handelt es sich bei den verwendeten Zelllinien nur um Modelle, die sich auf Grund der unterschiedlichen Genmuster von humanen Leberzellen oder Darmepithelzellen unterscheiden und nicht 1:1 auf den Menschen übertragen werden können.The primary objective of this work is to demonstrate in vitro that antiretroviral drugs can induce CYP3A4 mRNA in cell cultures. In addition, the CYP3A4 expression in various cell cultures was examined by determination of the CYP3A4-mRNA in these cultures. For the series of exper-iments, the cell lines HeLa, HepG2, Caco-2, Colo320 and human liver cells were tested for their content of CYP3A4-mRNA. The incubation experiments were carried out using the cell lines HepG2 and Colo320. We tested the antiretroviral drugs abacavir, amprenavir, zidovudine, dida-nosine, stavudine, saquinavir, zalcitabine, efavirenz, nevirapine, delavirdine, indinavir, lop-inavir/ritonavir and nelfinavir. As a reference substance for the induction the well known CYP3A4-inducer rifampicin was used. The incubation experiments were carried out in cell cul-ture 6-well-plates. Each well contained approximately one million cells. The Cells were treated with the test substances in various concentrations. After an incubation period of 20 hours at 37°C, the total RNA was isolated from the cells and converted to cDNA. Quantifying the CYP3A4 mRNA was carried out by using real-time PCR with the LightCycler-System (Roche). An external standard that was identical in sequence to the measured CYP3A4 mRNA was used to quantify the mRNA amount. The investigated nucleoside reverse transcriptase inhibitors (abacavir, didanosine, lamivudine, stavudine, zalcitabine, and zidovudine) did not induce the formation of CYP3A4-mRNA in the investigated cell lines. In the group of non-nucleoside reverse transcriptase inhibitors, a signifi-cant induction of CYP3A4 expression could be demonstrated for the substances efavirenz and delavirdine, but not for nevirapine. For the investigated protease inhibitors amprenavir, indinavir, nelfinavir, ritonavir and saquinavir induction of CYP3A4 expression in the cell lines HepG2 and Colo320 could be shown. The compound of the protease inhibitor Kaletra ® (lopinavir/ritonavir) did not induce the CYP3A4 gene expression in HepG2 cells but it did in Colo320 cells. The pre-sented experiments demonstrate that the CYP3A4-mRNA content decreases in the cells of the cell line HepG2, with increasing passage number. It was also demonstrated that the cell lines HepG2 and Colo320 contain lower amounts of CYP3A4 mRNA than cells of human liver tissue. With this study it was possible at the time of the experiments to prove an inductive power for the investigated protease inhibitors and some of the non-nucleoside reverse transcriptase inhibitors. Despite the well known inhibitory effect of protease inhibitors in vivo, these study present in-vitro an increase of CYP3A4-mRNA in cell-lines

Comprehensive systematic review and meta-analysis on physical health conditions in lesbian- and bisexual-identified women compared with heterosexual-identified women

Background: Sexual minority individuals experience discrimination, leading to mental health disparities. Physical health disparities have not been examined to the same extent in systematic reviews so far. Objectives: To provide a systematic review and, where possible, meta-analyses on the prevalence of physical health conditions in sexual minority women (i.e. lesbian- and bisexual-identified women) compared to heterosexual-identified women. Design: The study design is a systematic review with meta-analyses. Data Sources and Methods: A systematic literature search in MEDLINE, EMBASE, CENTRAL, CINAHL, and Web of Science databases was conducted on epidemiologic studies on physical health conditions, classified in the Global Burden of Disease project, published between 2000 and 2021. Meta-analyses pooling odds ratios were calculated. Results: In total, 23,649 abstracts were screened and 44 studies were included in the systematic review. Meta-analyses were run for arthritis, asthma, back pain, cancer, chronic kidney diseases, diabetes, headache disorders, heart attacks, hepatitis, hypertension, and stroke. Most significant differences in prevalence by sexual identity were found for chronic respiratory conditions, especially asthma. Overall, sexual minority women were significantly 1.5–2 times more likely to have asthma than heterosexual women. Furthermore, evidence of higher prevalence in sexual minority compared to heterosexual women was found for back pain, headaches/migraines, hepatitis B/C, periodontitis, urinary tract infections, and acne. In contrast, bisexual women had lower cancer rates. Overall, sexual minority women had lower odds of heart attacks, diabetes, and hypertension than heterosexual women (in terms of diabetes and hypertension possibly due to non-consideration of pregnancy-related conditions). Conclusion: We found evidence for physical health disparities by sexual identity. Since some of these findings rely on few comparisons only, this review emphasizes the need for routinely including sexual identity assessment in health research and clinical practice. Providing a more detailed picture of the prevalence of physical health conditions in sexual minority women may ultimately contribute to reducing health disparities

Effects of Resistance Training on Motor- and Non-Motor Symptoms in Patients with Parkinson's Disease: A Systematic Review and Meta-Analysis

Background: Previous reviews indicated positive effects of resistance training (RT) on motor outcomes in Parkinson's disease (PD). However, inconsistencies between the included studies exist, and non-motor outcomes have only scarcely been considered in a review on RT in PD. Objective: To analyze the RT effects on motor- and non-motor outcomes in PD patients compared to passive and physically active control groups (i.e., other structured physical interventions). Methods: We searched CENTRAL, MEDLINE, EMBASE, and CINAHL for randomized controlled trials of RT in PD. After identifying 18 studies, a meta-analysis was conducted for the outcomes muscle strength, motor impairment, freezing of gait (FoG), mobility and balance, quality of life (QoL), depression, cognition, and adverse events. Meta-analyses with random models were calculated using mean differences (MD) or standardized mean differences (SMD) with 95% confidence intervals (CI). Results: When comparing RT with passive control groups, the meta-analyses showed significant large effects on muscle strength (SMD = -0.84, 95% CI -1.29--0.39, p = 0.0003), motor impairment (SMD = -0.81, 95% CI -1.34--0.27, p = 0.003), mobility and balance (MD = -1.81, 95% CI -3.13-0.49, p = 0.007), and small significant effects on QoL (SMD = -0.48, 95% CI -0.86-0.10, p = 0.01). RT compared with physically active control groups reached no significant results for any outcome. Conclusion: RT improves muscle strength, motor impairment, mobility and balance, QoL, and depression in PD patients. However, it is not superior to other physically active interventions. Therefore, exercise is important for PD patients but according to this analysis, its type is of secondary interest

Measuring parathyroid hormone (PTH) in patients with oxidative stress--do we need a fourth generation parathyroid hormone assay?

Oxidation of PTH at methionine residues results in loss of biological activity. PTH may be oxidized in patients with renal disease. The aim of this study was to develop an assay considering oxidation of PTH. Oxidized hPTH was analyzed by high resolution nano-liquid chromatography coupled to ESI-FTT tandem mass spectrometry (nanoLC-ESI-FT-MS/MS) directly and after proteolytic cleavage. The oxidized hPTH(1-84) sample shows TIC-peaks at 18-20 min and several mass peaks due to mass shifts caused by oxidations. No significant signal for oxidized hPTH(1-84) species after removal of oxidized PTH molecules by a specific column with monoclonal antibodies (MAB) raised against the oxidized hPTH was detectable. By using this column in samples from 18 patients on dialysis we could demonstrate that measured PTH concentrations were substantially lower when considering oxidized forms of PTH. The relationship between PTH concentrations determined directly and those concentrations measured after removal of the oxidized PTH forms varies substantially. In some patients only 7% of traditionally measured PTH was free of oxidation, whereas in other patients 34% of the traditionally measured PTH was real intact PTH. In conclusion, a huge but not constant proportion of PTH molecules are oxidized in patients requiring dialysis. Since oxidized PTH is biologically inactive, the currently used methods to detect PTH in daily clinical practice may not adequately reflect PTH-related bone and cardiovascular abnormalities in patients on dialysis

Deduced molecular weights from the differently charged peaks in the spectra of the starting material, non-digested oxidized synthetic hPTH(1–84)ox (Fig. 1B), and the eluate from the affinity column (Fig. 3B).

<p>The molecular masses correspond to values shifted by +16, +32, +48, +64 Da caused by methionine oxidation (sulfoxide, +16 Da and sulfone, +32 Da for each residue, and combinations thereof, maximal+64 Da) and by +80 Da for the additional oxidation of tryptophan 23.</p

Comparison of the enlarged spectra of the starting material, non-digested oxidized synthetic hPTH(1–84)ox (<b>Fig.1B</b>), and the eluate from the affinity column of non-digested oxidized synthetic hPTH(1–84)ox (Fig. 3B).

<p>Comparison of the enlarged spectra of the starting material, non-digested oxidized synthetic hPTH(1–84)ox (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040242#pone-0040242-g001" target="_blank"><b>Fig.1B</b></a>), and the eluate from the affinity column of non-digested oxidized synthetic hPTH(1–84)ox (Fig. 3B).</p

Non-digested oxidized synthetic hPTH(1–84)ox.

<p>A: NanoLC-ESI-FTMS total ion chromatogram. B: Magnified summed FTMS spectrum for retention time interval 18.30–20.50 minutes. Several different charged analyte ions were detected.</p

Flow through fraction of non-digested oxidized synthetic hPTH(1–84)ox from the affinity column.

<p><b>A</b>: NanoLC-ESI-FTMS total ion chromatogram. <b>B</b>: Magnified summed FTMS spectrum for retention time interval of 16.50–18.50 minutes. The spectrum does not show any analyte masses which belong to PTH or oxidized PTH.</p