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Efflux transporters in rat placenta and developing brain: transcriptomic and functional response to paracetamol.

ABSTRACT: Adenosine triphosphate binding cassette (ABC) transporters transfer lipid-soluble molecules across cellular interfaces either directly or after enzymatic metabolism. RNAseq analysis identified transcripts for ABC transporters and enzymes in rat E19, P5 and adult brain and choroid plexus and E19 placenta. Their functional capacity to efflux small molecules was studied by quantitative analysis of paracetamol (acetaminophen) and its metabolites using liquid scintillation counting, autoradiography and ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC-MS/MS). Animals were treated acutely (30 min) and chronically (5 days, twice daily) with paracetamol (15 mg/kg) to investigate ability of brain and placenta barriers to regulate ABC transport functionality during extended treatment. Results indicated that transcripts of many efflux-associated ABC transporters were higher in adult brain and choroid plexus than at earlier ages. Chronic treatment upregulated certain transcripts only in adult brain and altered concentrations of paracetamol metabolites in circulation of pregnant dams. Combination of changes to metabolites and transport system transcripts may explain observed changes in paracetamol entry into adult and fetal brains. Analysis of lower paracetamol dosing (3.75 mg/kg) indicated dose-dependent changes in paracetamol metabolism. Transcripts of ABC transporters and enzymes at key barriers responsible for molecular transport into the developing brain showed alterations in paracetamol pharmacokinetics in pregnancy following different treatment regimens.

SUBMITTER: Koehn LM

PROVIDER: S-EPMC8494792 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background: Paracetamol (acetaminophen) is widely used in pregnancy and generally regarded as "safe" by regulatory authorities. Methods: Clinically relevant doses of paracetamol were administered intraperitoneally to pregnant rats twice daily from embryonic day E15 to 19 (chronic) or as a single dose at E19 (acute). Control samples were from un-treated age-matched animals. At E19, rats were anaesthetised, administered a final paracetamol dose, uteruses were opened and fetuses exposed for sample collection. For RNA sequencing, placentas and fetal brains were removed and flash frozen. Fetal and maternal plasma and cerebrospinal fluid were assayed for α-fetoprotein and interleukin 1β (IL1β). Brains were fixed and examined (immunohistochemistry) for plasma protein distribution. Placental permeability to a small molecule ( 14C-sucrose) was tested by injection into either mother or individual fetuses; fetal and maternal blood was sampled at regular intervals to 90 minutes. Results: RNA sequencing revealed a large number of genes up- or down-regulated in placentas from acutely or chronically treated animals compared to controls. Most notable was down-regulation of three acute phase plasma proteins (α-fetoprotein, transferrin, transthyretin) in acute and especially chronic experiments and marked up-regulation of immune-related genes, particularly cytokines, again especially in chronically treated dams. IL1β increased in plasma of most fetuses from treated dams but to variable levels and no IL1β was detectable in plasma of control fetuses or any of the dams. Increased placental permeability appeared to be only from fetus to mother for both 14C-sucrose and α-fetoprotein, but not in the reverse direction. In the fetal brain, gene regulatory changes were less prominent than in the placenta of treated fetuses and did not involve inflammatory-related genes; there was no evidence of increased blood-brain barrier permeability. Conclusion: Results suggest that paracetamol may induce an immune-inflammatory-like response in placenta and more caution should be exercised in use of paracetamol in pregnancy.

Project description:Toxicogenomics is a critical area of inquiry for hazard identification and to identify both mechanisms of action and potential markers of exposure to toxic compounds. However, data generated by these experiments are highly dimensional and present challenges to standard statistical approaches, requiring strict correction for multiple comparisons. This stringency often fails to detect meaningful changes to low expression genes and/or eliminate genes with small but consistent changes particularly in tissues where slight changes in expression can have important functional differences, such as brain. Machine learning offers an alternative analytical approach for "omics" data that effectively sidesteps the challenges of analyzing highly dimensional data. Using 3 rat RNA transcriptome sets, we utilized an ensemble machine learning approach to predict developmental exposure to a mixture of organophosphate esters (OPEs) in brain (newborn cortex and day 10 hippocampus) and late gestation placenta of male and female rats, and identified genes that informed predictor performance. OPE exposure had sex specific effects on hippocampal transcriptome, and significantly impacted genes associated with mitochondrial transcriptional regulation and cation transport in females, including voltage-gated potassium and calcium channels and subunits. To establish if this holds for other tissues, RNAseq data from cortex and placenta, both previously published and analyzed via a more traditional pipeline, were reanalyzed with the ensemble machine learning methodology. Significant enrichment for pathways of oxidative phosphorylation and electron transport chain was found, suggesting a transcriptomic signature of OPE exposure impacting mitochondrial metabolism across tissue types and developmental epoch. Here we show how machine learning can complement more traditional analytical approaches to identify vulnerable "signature" pathways disrupted by chemical exposures and biomarkers of exposure.

Project description:BackgroundPositron emission tomography (PET) is a molecular imaging technique that can be used to investigate the in vivo pharmacology of drugs. Initial preclinical evaluation of PET tracers is often conducted in rodents due to the accessibility of disease models as well as economic considerations. Compared to larger species, rodents display a higher expression and/or activity of efflux transporters such as the P-glycoprotein (P-gp). Low brain uptake could, therefore, be species-specific and uptake in rodents not be predictive for that in humans. We hypothesized that a better prediction from rodent data could be achieved when a tracer is evaluated under P-gp inhibition. Consequently, we compared the performance of eight neuroreceptor tracers in rats with and without P-gp inhibition including a specific binding blockade. This data set was then used to predict the binding of these eight tracers in pigs.MethodsPET tracers targeting serotonin 5-HT2A receptors ([18F]MH.MZ, [18F]Altanserin, [11C]Cimbi-36, [11C]Pimavanserin), serotonin 5-HT7 receptors ([11C]Cimbi-701, [11C]Cimbi-717 and [11C]BA-10) and dopamine D2/3 receptors ([18F]Fallypride) were used in the study. The brain uptake and target-specific binding of these PET radiotracers were evaluated in rats with and without inhibition of P-gp. Rat data were subsequently compared to the results obtained in pigs.ResultsWithout P-gp inhibition, the amount of target-specific binding in the rat brain was sufficient to justify further translation for three out of eight evaluated tracers. With P-gp inhibition, results for five out of eight tracers justified further translation. The performance in pigs could correctly be predicted for six out of eight tracers when rat data obtained under P-gp inhibition were used, compared to four out of eight tracers without P-gp inhibition.ConclusionsP-gp strongly affects the uptake of PET tracers in rodents, but false prediction outcomes can be reduced by evaluating a tracer under P-gp inhibition.

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Efflux transporters in rat placenta and developing brain: transcriptomic and functional response to paracetamol.

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