Practical Semiquantification Strategy for Estimating Suspect Per- and Polyfluoroalkyl Substance (PFAS) Concentrations

Semiquantitation of suspect per- and polyfluoroalkyl substances (PFAS) in complex mixtures is challenging due to the increasing number of suspect PFAS. Traditional 1:1 matching strategies require selecting calibrants (target–surrogate standard pairs) based on head group, fluorinated chain length, and retention time, which is time-consuming and requires expert knowledge. Lack of uniformity in calibrant selection for estimating suspect concentrations among different laboratories makes comparing reported suspect concentrations difficult. In this study, a practical approach whereby the area counts for 50 anionic and 5 zwitterionic/cationic target PFAS were ratioed to the average area of their respective stable-isotope labeled surrogates to create “average PFAS calibration curves” for suspects detected in negative- and positive-ionization mode liquid chromatography quadrupole time-of-flight mass spectrometry. The calibration curves were fitted with log–log and weighted linear regression models. The two models were evaluated for their accuracy and prediction interval in predicting the target PFAS concentrations. The average PFAS calibration curves were then used to estimate the suspect PFAS concentration in a well-characterized aqueous film-forming foam. Weighted linear regression resulted in more target PFAS that fell within 70–130% of their known standard value and narrower prediction intervals than the log–log transformation approach. The summed suspect PFAS concentrations calculated by weighted linear regression and log–log transformation were within 8 and 16% of those estimated by a 1:1 matching strategy. The average PFAS calibration curve can be easily expanded and can be applied to any suspect PFAS even if the confidence in the suspect structure is low or unknown

Practical Semiquantification Strategy for Estimating Suspect Per- and Polyfluoroalkyl Substance (PFAS) Concentrations

Semiquantitation of suspect per- and polyfluoroalkyl substances (PFAS) in complex mixtures is challenging due to the increasing number of suspect PFAS. Traditional 1:1 matching strategies require selecting calibrants (target–surrogate standard pairs) based on head group, fluorinated chain length, and retention time, which is time-consuming and requires expert knowledge. Lack of uniformity in calibrant selection for estimating suspect concentrations among different laboratories makes comparing reported suspect concentrations difficult. In this study, a practical approach whereby the area counts for 50 anionic and 5 zwitterionic/cationic target PFAS were ratioed to the average area of their respective stable-isotope labeled surrogates to create “average PFAS calibration curves” for suspects detected in negative- and positive-ionization mode liquid chromatography quadrupole time-of-flight mass spectrometry. The calibration curves were fitted with log–log and weighted linear regression models. The two models were evaluated for their accuracy and prediction interval in predicting the target PFAS concentrations. The average PFAS calibration curves were then used to estimate the suspect PFAS concentration in a well-characterized aqueous film-forming foam. Weighted linear regression resulted in more target PFAS that fell within 70–130% of their known standard value and narrower prediction intervals than the log–log transformation approach. The summed suspect PFAS concentrations calculated by weighted linear regression and log–log transformation were within 8 and 16% of those estimated by a 1:1 matching strategy. The average PFAS calibration curve can be easily expanded and can be applied to any suspect PFAS even if the confidence in the suspect structure is low or unknown

Table5_Activation of the PPARγ Prevents Ferroptosis-Induced Neuronal Loss in Response to Intracerebral Hemorrhage Through Synergistic Actions With the Nrf2.XLSX

Intracerebral hemorrhage (ICH) is a subtype of stroke characterized by high mortality and disability rates. The long-term effects of ICH-induced intracranial hematoma on patients’ neurological function are unclear. Currently, an effective treatment that significantly reduces the rates of death and disability in patients with ICH is not available. Based on accumulating evidence, ferroptosis may be the leading factor contributing to the neurological impairment caused by ICH injury. Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated receptor in the nuclear hormone receptor family that synergistically interacts with the nuclear factor erythrocyte 2-related factor 2 (Nrf2) pathway to promote the expression of related genes and inhibit ferroptosis. Primary rat hippocampal neurons were treated with heme (50 μM) and erastin (50 μM) to induce ferroptosis, followed by the PPARγ agonist pioglitazone (PDZ, 10 μM) to verify the inhibitory effect of PPARγ activation on ferroptosis. ML385 (2 μM), a novel and specific NRF2 inhibitor, was administered to the inhibitor group, followed by an analysis of cellular activity and immunofluorescence staining. In vivo Assays, ICH rats injected with autologous striatum were treated with 30 mg/kg/d pioglitazone, and the inhibitor group was injected with ML385 (30 mg/kg). The results showed that PDZ inhibited ferroptosis in neurons by increasing the expression of PPARγ, Nrf2 and Gpx4 in vitro, while PDZ reduced ferroptosis in neurons after ICH and promoted the recovery of neural function in vivo. Our results suggest that PDZ, a PPARγ agonist, promotes Gpx4 expression through the interaction between PPARγ and the Nrf2 pathway, inhibits ferroptosis of neurons after ICH, and promotes the recovery of neural function.</p

Table2_Curcumin Restrains Oxidative Stress of After Intracerebral Hemorrhage in Rat by Activating the Nrf2/HO-1 Pathway.XLSX

Intracerebral hemorrhage (ICH), a severe hemorrhagic stroke, induces cerebral oxidative stress and severe secondary neurological injury. Curcumin was demonstrated to inhibit oxidative stress in the brain after ICH. However, the pharmacological mechanism needs further research. We used an intrastriatal injection of autologous blood to make the rat ICH model, and then the rat was treated with curcumin (100 mg/kg/day). Modified Neurological Severity Score (mNSS) and corner test results showed that curcumin could significantly promote the neurological recovery of ICH rats. Meanwhile, curcumin could substantially reduce ROS and MDA in the tissues around intracranial hematoma and prevent GSH depletion. To explore the pharmacological molecular mechanism of curcumin, we used HAPI cells and primary rat cortical microglia for in vitro experiments. In vitro, heme-treated cells were used as the cell model of ICH to explore the molecular mechanism of inhibiting oxidative stress by curcumin treatment. The results showed that curcumin significantly inhibited heme-induced oxidative stress, decreased intracellular ROS and MDA, and promoted Nrf2 and its downstream antioxidant gene (HO-1, NQO1, and Gpx4) expression. These results suggest that curcumin inhibits oxidative stress by activating the Nrf2/HO-1 pathway. Here, our results indicate that curcumin can promote the inhibition of oxidative stress in microglia by activating the Nrf2/HO-1 pathway and promoting neurological recovery after ICH, providing a new therapeutic target for clinical treatment of ICH.</p

Table7_Activation of the PPARγ Prevents Ferroptosis-Induced Neuronal Loss in Response to Intracerebral Hemorrhage Through Synergistic Actions With the Nrf2.XLSX

Intracerebral hemorrhage (ICH) is a subtype of stroke characterized by high mortality and disability rates. The long-term effects of ICH-induced intracranial hematoma on patients’ neurological function are unclear. Currently, an effective treatment that significantly reduces the rates of death and disability in patients with ICH is not available. Based on accumulating evidence, ferroptosis may be the leading factor contributing to the neurological impairment caused by ICH injury. Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated receptor in the nuclear hormone receptor family that synergistically interacts with the nuclear factor erythrocyte 2-related factor 2 (Nrf2) pathway to promote the expression of related genes and inhibit ferroptosis. Primary rat hippocampal neurons were treated with heme (50 μM) and erastin (50 μM) to induce ferroptosis, followed by the PPARγ agonist pioglitazone (PDZ, 10 μM) to verify the inhibitory effect of PPARγ activation on ferroptosis. ML385 (2 μM), a novel and specific NRF2 inhibitor, was administered to the inhibitor group, followed by an analysis of cellular activity and immunofluorescence staining. In vivo Assays, ICH rats injected with autologous striatum were treated with 30 mg/kg/d pioglitazone, and the inhibitor group was injected with ML385 (30 mg/kg). The results showed that PDZ inhibited ferroptosis in neurons by increasing the expression of PPARγ, Nrf2 and Gpx4 in vitro, while PDZ reduced ferroptosis in neurons after ICH and promoted the recovery of neural function in vivo. Our results suggest that PDZ, a PPARγ agonist, promotes Gpx4 expression through the interaction between PPARγ and the Nrf2 pathway, inhibits ferroptosis of neurons after ICH, and promotes the recovery of neural function.</p

Image5_Activation of the PPARγ Prevents Ferroptosis-Induced Neuronal Loss in Response to Intracerebral Hemorrhage Through Synergistic Actions With the Nrf2.TIF

Intracerebral hemorrhage (ICH) is a subtype of stroke characterized by high mortality and disability rates. The long-term effects of ICH-induced intracranial hematoma on patients’ neurological function are unclear. Currently, an effective treatment that significantly reduces the rates of death and disability in patients with ICH is not available. Based on accumulating evidence, ferroptosis may be the leading factor contributing to the neurological impairment caused by ICH injury. Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated receptor in the nuclear hormone receptor family that synergistically interacts with the nuclear factor erythrocyte 2-related factor 2 (Nrf2) pathway to promote the expression of related genes and inhibit ferroptosis. Primary rat hippocampal neurons were treated with heme (50 μM) and erastin (50 μM) to induce ferroptosis, followed by the PPARγ agonist pioglitazone (PDZ, 10 μM) to verify the inhibitory effect of PPARγ activation on ferroptosis. ML385 (2 μM), a novel and specific NRF2 inhibitor, was administered to the inhibitor group, followed by an analysis of cellular activity and immunofluorescence staining. In vivo Assays, ICH rats injected with autologous striatum were treated with 30 mg/kg/d pioglitazone, and the inhibitor group was injected with ML385 (30 mg/kg). The results showed that PDZ inhibited ferroptosis in neurons by increasing the expression of PPARγ, Nrf2 and Gpx4 in vitro, while PDZ reduced ferroptosis in neurons after ICH and promoted the recovery of neural function in vivo. Our results suggest that PDZ, a PPARγ agonist, promotes Gpx4 expression through the interaction between PPARγ and the Nrf2 pathway, inhibits ferroptosis of neurons after ICH, and promotes the recovery of neural function.</p

Table6_Curcumin Restrains Oxidative Stress of After Intracerebral Hemorrhage in Rat by Activating the Nrf2/HO-1 Pathway.XLSX

Intracerebral hemorrhage (ICH), a severe hemorrhagic stroke, induces cerebral oxidative stress and severe secondary neurological injury. Curcumin was demonstrated to inhibit oxidative stress in the brain after ICH. However, the pharmacological mechanism needs further research. We used an intrastriatal injection of autologous blood to make the rat ICH model, and then the rat was treated with curcumin (100 mg/kg/day). Modified Neurological Severity Score (mNSS) and corner test results showed that curcumin could significantly promote the neurological recovery of ICH rats. Meanwhile, curcumin could substantially reduce ROS and MDA in the tissues around intracranial hematoma and prevent GSH depletion. To explore the pharmacological molecular mechanism of curcumin, we used HAPI cells and primary rat cortical microglia for in vitro experiments. In vitro, heme-treated cells were used as the cell model of ICH to explore the molecular mechanism of inhibiting oxidative stress by curcumin treatment. The results showed that curcumin significantly inhibited heme-induced oxidative stress, decreased intracellular ROS and MDA, and promoted Nrf2 and its downstream antioxidant gene (HO-1, NQO1, and Gpx4) expression. These results suggest that curcumin inhibits oxidative stress by activating the Nrf2/HO-1 pathway. Here, our results indicate that curcumin can promote the inhibition of oxidative stress in microglia by activating the Nrf2/HO-1 pathway and promoting neurological recovery after ICH, providing a new therapeutic target for clinical treatment of ICH.</p

Image4_Activation of the PPARγ Prevents Ferroptosis-Induced Neuronal Loss in Response to Intracerebral Hemorrhage Through Synergistic Actions With the Nrf2.JPEG

Intracerebral hemorrhage (ICH) is a subtype of stroke characterized by high mortality and disability rates. The long-term effects of ICH-induced intracranial hematoma on patients’ neurological function are unclear. Currently, an effective treatment that significantly reduces the rates of death and disability in patients with ICH is not available. Based on accumulating evidence, ferroptosis may be the leading factor contributing to the neurological impairment caused by ICH injury. Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated receptor in the nuclear hormone receptor family that synergistically interacts with the nuclear factor erythrocyte 2-related factor 2 (Nrf2) pathway to promote the expression of related genes and inhibit ferroptosis. Primary rat hippocampal neurons were treated with heme (50 μM) and erastin (50 μM) to induce ferroptosis, followed by the PPARγ agonist pioglitazone (PDZ, 10 μM) to verify the inhibitory effect of PPARγ activation on ferroptosis. ML385 (2 μM), a novel and specific NRF2 inhibitor, was administered to the inhibitor group, followed by an analysis of cellular activity and immunofluorescence staining. In vivo Assays, ICH rats injected with autologous striatum were treated with 30 mg/kg/d pioglitazone, and the inhibitor group was injected with ML385 (30 mg/kg). The results showed that PDZ inhibited ferroptosis in neurons by increasing the expression of PPARγ, Nrf2 and Gpx4 in vitro, while PDZ reduced ferroptosis in neurons after ICH and promoted the recovery of neural function in vivo. Our results suggest that PDZ, a PPARγ agonist, promotes Gpx4 expression through the interaction between PPARγ and the Nrf2 pathway, inhibits ferroptosis of neurons after ICH, and promotes the recovery of neural function.</p

Table9_Activation of the PPARγ Prevents Ferroptosis-Induced Neuronal Loss in Response to Intracerebral Hemorrhage Through Synergistic Actions With the Nrf2.XLSX

Intracerebral hemorrhage (ICH) is a subtype of stroke characterized by high mortality and disability rates. The long-term effects of ICH-induced intracranial hematoma on patients’ neurological function are unclear. Currently, an effective treatment that significantly reduces the rates of death and disability in patients with ICH is not available. Based on accumulating evidence, ferroptosis may be the leading factor contributing to the neurological impairment caused by ICH injury. Peroxisome proliferator-activated receptor γ (PPARγ) is a ligand-activated receptor in the nuclear hormone receptor family that synergistically interacts with the nuclear factor erythrocyte 2-related factor 2 (Nrf2) pathway to promote the expression of related genes and inhibit ferroptosis. Primary rat hippocampal neurons were treated with heme (50 μM) and erastin (50 μM) to induce ferroptosis, followed by the PPARγ agonist pioglitazone (PDZ, 10 μM) to verify the inhibitory effect of PPARγ activation on ferroptosis. ML385 (2 μM), a novel and specific NRF2 inhibitor, was administered to the inhibitor group, followed by an analysis of cellular activity and immunofluorescence staining. In vivo Assays, ICH rats injected with autologous striatum were treated with 30 mg/kg/d pioglitazone, and the inhibitor group was injected with ML385 (30 mg/kg). The results showed that PDZ inhibited ferroptosis in neurons by increasing the expression of PPARγ, Nrf2 and Gpx4 in vitro, while PDZ reduced ferroptosis in neurons after ICH and promoted the recovery of neural function in vivo. Our results suggest that PDZ, a PPARγ agonist, promotes Gpx4 expression through the interaction between PPARγ and the Nrf2 pathway, inhibits ferroptosis of neurons after ICH, and promotes the recovery of neural function.</p

Table4_Curcumin Restrains Oxidative Stress of After Intracerebral Hemorrhage in Rat by Activating the Nrf2/HO-1 Pathway.XLSX

Intracerebral hemorrhage (ICH), a severe hemorrhagic stroke, induces cerebral oxidative stress and severe secondary neurological injury. Curcumin was demonstrated to inhibit oxidative stress in the brain after ICH. However, the pharmacological mechanism needs further research. We used an intrastriatal injection of autologous blood to make the rat ICH model, and then the rat was treated with curcumin (100 mg/kg/day). Modified Neurological Severity Score (mNSS) and corner test results showed that curcumin could significantly promote the neurological recovery of ICH rats. Meanwhile, curcumin could substantially reduce ROS and MDA in the tissues around intracranial hematoma and prevent GSH depletion. To explore the pharmacological molecular mechanism of curcumin, we used HAPI cells and primary rat cortical microglia for in vitro experiments. In vitro, heme-treated cells were used as the cell model of ICH to explore the molecular mechanism of inhibiting oxidative stress by curcumin treatment. The results showed that curcumin significantly inhibited heme-induced oxidative stress, decreased intracellular ROS and MDA, and promoted Nrf2 and its downstream antioxidant gene (HO-1, NQO1, and Gpx4) expression. These results suggest that curcumin inhibits oxidative stress by activating the Nrf2/HO-1 pathway. Here, our results indicate that curcumin can promote the inhibition of oxidative stress in microglia by activating the Nrf2/HO-1 pathway and promoting neurological recovery after ICH, providing a new therapeutic target for clinical treatment of ICH.</p