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BackgroundOxidative stress from excess reactive oxygen species (ROS) is a hypothesized contributor to preterm birth. Per- and polyfluoroalkyl substances (PFAS) exposure is reported to generate ROS in laboratory settings, and is linked to adverse birth outcomes globally. However, to our knowledge, the relationship between PFAS and oxidative stress has not been examined in the context of human pregnancy.ObjectiveTo investigate the associations between prenatal PFAS exposure and oxidative stress biomarkers among pregnant people.MethodsOur analytic sample included 428 participants enrolled in the Illinois Kids Development Study and Chemicals In Our Bodies prospective birth cohorts between 2014 and 2019. Twelve PFAS were measured in second trimester serum. We focused on seven PFAS that were detected in>65% of participants. Urinary levels of 8-isoprostane-prostaglandin-F2α, prostaglandin-F2α, 2,3-dinor-8-iso-PGF2α, and 2,3-dinor-5,6-dihydro-8-iso-PGF2α were measured in the second and third trimesters as biomarkers of oxidative stress. We fit linear mixed-effects models to estimate individual associations between PFAS and oxidative stress biomarkers. We used quantile g-computation and Bayesian kernel machine regression (BKMR) to assess associations between the PFAS mixture and averaged oxidative stress biomarkers.ResultsLinear mixed-effects models showed that an interquartile range increase in perfluorooctane sulfonic acid (PFOS) was associated with an increase in 8-isoprostane-prostaglandin-F2α (β=0.10, 95% confidence interval=0, 0.20). In both quantile g-computation and BKMR, and across all oxidative stress biomarkers, PFOS contributed the most to the overall mixture effect. The six remaining PFAS were not significantly associated with changes in oxidative stress biomarkers.ConclusionsOur study is the first to investigate the relationship between PFAS exposure and biomarkers of oxidative stress during human pregnancy. We found that PFOS was associated with elevated levels of oxidative stress, which is consistent with prior work in animal models and cell lines. Future research is needed to understand how prenatal PFAS exposure and maternal oxidative stress may affect fetal development.

Cannabinoid receptors (CBR) are important drug targets for the treatment of various inflammatory, metabolic and neurological diseases. Therefore, sensitive test systems for the assessment of ligands are needed. In this study, a steady-state GTPase assay for human CBR subtypes 1 and 2 was developed to characterize the pharmacological property of ligands at a very proximal point of the signal transduction cascade. Establishing these in vitro test sytems, we studied cell or tissue membranes heterogenously or endogenously expressing CBR, such as CBR-infected Human Embryonic Kidney (HEK) 293 cells, rat cerebellum and spleen cells. The lack of effects in the GTPase assay and in [(35)S]GTPgammaS binding experiments in these expression system, directed us to use Spodoptera frugiperda (Sf9) cells. Co-expressing CBR, different Galpha-subunits, Gbetagamma heterodimer, and RGS (Regulator of G-protein signaling)-proteins in Sf9 cell membranes greatly improved the sensitivity of the assay, with highest GTPase activation in the CBR + Galpha(i2) + Gbeta(1)gamma(2) + RGS4 system. We examined exogenous and endogenous standard ligands as well as secondary metabolites as Delta(9)-tetrahydrocannabinol (Delta(9)-THC), dodeca-2E,4E-dienoic acid isobutylamide, an alkylamide from Echinacea purpurea, and an E. purpurea hexane extract according their agonistic and antagonistic properties. The suitability of the assay for screening procedures was also proven by detecting the activity of Delta(9)-THC in a matrix of other less active compounds (Delta(9)-THC-free Cannabis sativa extract). In conclusion, we have developed highly sensitive test systems for the analysis of CBR ligands.

Human cannabinoid receptors 1 (hCB(1)R) and 2 (hCB(2)R) are expressed in the CNS and couple to G(i)/G(o)-proteins. The aim of this study was to compare coupling of hCB(1)R and hCB(2)R to G(alpha)(i2)beta(1)gamma(2) in Sf9 insect cells. High-affinity agonist binding at hCB(1)R, but not at hCB(2)R, was resistant to guanine nucleotides. hCB(1)R activated G(alpha)(i2)beta(1)gamma(2) much more rapidly than hCB(2)R in the [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTPgammaS) binding assay. Moreover, hCB(1)R exhibited a higher constitutive activity than hCB(2)R as assessed by the relative inhibitory effects of inverse agonists on [(35)S]GTPgammaS binding and steady-state high-affinity GTPase activity compared to the stimulatory effects of the hCB(1/2)R agonist CP 55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol]. G(alpha)(i2)beta(1)gamma(2) coupled to hCB(2)R exhibited higher GDP- and GTPgammaS-affinities than G(alpha)(i2)beta(1)gamma(2) coupled to hCB(1)R. NaCl effectively reduced constitutive activity of hCB(1)R but not of hCB(2)R. Collectively, hCB(1)R and hCB(2)R couple differentially to G(alpha)(i2)beta(1)gamma(2). Moreover, hCB(1)R exhibits higher constitutive activity than hCB(2)R. These differences point to distinct functions of hCB(1)R and hCB(2)R in the CNS.