Loss of flavin-containing monooxygenase 3 modulates dioxin-like polychlorinated biphenyl 126-induced oxidative stress and hepatotoxicity.

Dioxin-like pollutants (DLPs), such as polychlorinated biphenyl 126 (PCB 126), are synthetic chemicals classified as persistent organic pollutants. They accumulate in adipose tissue and have been linked to cardiometabolic disorders, including fatty liver disease. The toxicity of these compounds is associated with activation of the aryl hydrocarbon receptor (Ahr), leading to the induction of phase I metabolizing enzyme cytochrome P4501a1 (Cyp1a1) and the subsequent production of reactive oxygen species (ROS). Recent research has shown that DLPs can also induce the xenobiotic detoxification enzyme flavin-containing monooxygenase 3 (FMO3), which plays a role in metabolic homeostasis. We hypothesized whether genetic deletion of Fmo3 could protect mice, particularly in the liver, where Fmo3 is most inducible, against PCB 126 toxicity. To test this hypothesis, male C57BL/6 wild-type (WT) mice and Fmo3 knockout (Fmo3 KO) mice were exposed to PCB 126 or vehicle (safflower oil) during a 12-week study, at weeks 2 and 4. Various analyses were performed, including hepatic histology, RNA-sequencing, and quantitation of PCB 126 and F2-isoprostane concentrations. The results showed that PCB 126 exposure caused macro and microvesicular fat deposition in WT mice, but this macrovesicular fatty change was absent in Fmo3 KO mice. Moreover, at the pathway level, the hepatic oxidative stress response was significantly different between the two genotypes, with the induction of specific genes observed only in WT mice. Notably, the most abundant F2-isoprostane, 8-iso-15-keto PGE2, increased in WT mice in response to PCB 126 exposure. The study's findings also demonstrated that hepatic tissue concentrations of PCB 126 were higher in WT mice compared to Fmo3 KO mice. In summary, the absence of FMO3 in mice led to a distinctive response to dioxin-like pollutant exposure in the liver, likely due to alterations in lipid metabolism and storage, underscoring the complex interplay of genetic factors in the response to environmental toxins.

Exposure to a mixture of per-and polyfluoroalkyl substances modulates pulmonary expression of ACE2 and circulating hormones and cytokines.

Genetic and environmental factors impact on the interindividual variability of susceptibility to communicable and non-communicable diseases. A class of ubiquitous chemicals, Per- and polyfluoroalkyl substances (PFAS) have been linked in epidemiological studies to immunosuppression and increased susceptibility to viral infections, but possible mechanisms are not well elucidated. To begin to gain insight into the role of PFAS in susceptibility to one such viral infection, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), male and female C57BL/6 J mice were exposed to control water or a mixture of 5 PFAS (PFOS, PFOA, PFNA, PFHxS, Genx) for 12 weeks and lungs were isolated for examination of expression of SARS-CoV-2-related receptors Angiotensin-Converting Enzyme 2 (ACE2) and others. Secondary analyses included circulating hormones and cytokines which have been shown to directly or indirectly impact on ACE2 expression and severity of viral infections. Changes in mRNA and protein expression were analyzed by RT-qPCR and western blotting and circulating hormones and cytokines were determined by ELISA and MESO QuickPlex. The PFAS mixture decreased Ace2 mRNA 2.5-fold in male mice (p < 0.0001), with no significant change observed in females. In addition, TMPRSS2, ANPEP, ENPEP and DPP4 (other genes implicated in COVID-19 infection) were modulated due to PFAS. Plasma testosterone, but not estrogen were strikingly decreased due to PFAS which corresponded to PFAS-mediated repression of 4 representative pulmonary AR target genes; hemoglobin, beta adult major chain (Hbb-b1), Ferrochelatase (Fech), Collagen Type XIV Alpha 1 Chain (Col14a1), 5'-Aminolevulinate Synthase 2 (Alas2). Finally, PFAS modulated circulating pro and anti-inflammatory mediators including IFN-γ (downregulated 3.0-fold in females; p = 0.0301, 2.1-fold in males; p = 0.0418) and IL-6 (upregulated 5.6-fold in males; p = 0.030, no change in females). In conclusion, our data indicate long term exposure to a PFAS mixture impacts mechanisms related to expression of ACE2 in the lung. This work provides a mechanistic rationale for important future studies of PFAS exposure and subsequent viral infection.

Dichotomous Role of Plasmin in Regulation of Macrophage Function after Acetaminophen Overdose.

Kupffer cells and monocyte-derived macrophages are critical for liver repair after acetaminophen (APAP) overdose. These cells produce promitogenic cytokines and growth factors, and they phagocytose dead cell debris, a process that is critical for resolution of inflammation. The factors that regulate these dynamic functions of macrophages after APAP overdose, however, are not fully understood. We tested the hypothesis that the fibrinolytic enzyme, plasmin, is a key regulator of macrophage function after APAP-induced liver injury. In these studies, inhibition of plasmin in mice with tranexamic acid delayed up-regulation of proinflammatory cytokines after APAP overdose. In culture, plasmin directly, and in synergy with high-mobility group B1, stimulated Kupffer cells and bone marrow-derived macrophages to produce cytokines by a mechanism that required NF-κB. Inhibition of plasmin in vivo also prevented trafficking of monocyte-derived macrophages into necrotic lesions after APAP overdose. This prevented phagocytic removal of dead cells, prevented maturation of monocyte-derived macrophages into F4/80-expressing macrophages, and prevented termination of proinflammatory cytokine production. Our studies reveal further that phagocytosis is an important stimulus for cessation of proinflammatory cytokine production as treatment of proinflammatory, monocyte-derived macrophages, isolated from APAP-treated mice, with necrotic hepatocytes decreased expression of proinflammatory cytokines. Collectively, these studies demonstrate that plasmin is an important regulator of macrophage function after APAP overdose.

Patient perspectives on ipilimumab across the melanoma treatment trajectory.

PURPOSE: Ipilimumab was the first FDA-approved agent for advanced melanoma to improve survival and represents a paradigm shift in melanoma and cancer treatment. Its unique toxicity profile and kinetics of treatment response raise novel patient education challenges. We assessed patient perceptions of ipilimumab therapy across the treatment trajectory. METHODS: Four patient cohorts were assessed at different time points relative to treatment initiation: (1) prior to initiation of ipilimumab (n = 10), (2) at weeks 10-12 before restaging studies (n = 11), (3) at week 12 following restaging studies indicating progression of disease (n = 10), and (4) at week 12 following restaging studies indicating either a radiographic response or disease stability (n = 10). Patients participated in a semistructured qualitative interview to assess their experiences with ipilimumab. Quality of life was assessed via the Functional Assessment of Cancer Therapy-General and its Melanoma-specific module. RESULTS: Perceived quality of life was comparable across cohorts, and a majority of the sample understood side effects from ipilimumab and the potential for a delayed treatment response. Patients without progression of disease following restaging studies at week 12 held more positive views regarding ipilimumab compared to patients who had progressed. CONCLUSION: Patients generally regarded ipilimumab positively despite the risk of unique toxicities and potential for delayed therapeutic responses; however, those with progression expressed uncertainty regarding whether taking ipilimumab was worthwhile. Physician communication practices and patient education regarding realistic expectations for therapeutic benefit as well as unique toxicities associated with ipilimumab should be developed so that patients can better understand the possible outcomes from treatment.

Hepatic stellate cells orchestrate clearance of necrotic cells in a hypoxia-inducible factor-1α-dependent manner by modulating macrophage phenotype in mice.

Hypoxia-inducible factor-1α (HIF-1α) is activated in hepatic stellate cells (HSCs) by hypoxia and regulates genes important for tissue repair. Whether HIF-1α is activated in HSCs after acute injury and contributes to liver regeneration, however, is not known. To investigate this, mice were generated with reduced levels of HIF-1α in HSCs by crossing HIF-1α floxed mice with mice that express Cre recombinase under control of the glial fibrillary acidic protein (GFAP) promoter (i.e., HIF-1α-GFAP Cre+ mice). These mice and control mice (i.e., HIF-1α-GFAP Cre- mice) were treated with a single dose of carbon tetrachloride, and liver injury and repair were assessed. After carbon tetrachloride, HIF-1α was activated in HSCs. Although liver injury was not different between the two strains of mice, during resolution of injury, clearance of necrotic cells was decreased in HIF-1α-GFAP Cre+ mice. In these mice, the persistence of necrotic cells stimulated a fibrotic response characterized by extensive collagen deposition. Hepatic accumulation of macrophages, which clear necrotic cells from the liver after carbon tetrachloride, was not affected by HIF-1α deletion in HSCs. Conversion of macrophages to M1-like, proinflammatory macrophages, which have increased phagocytic activity, however, was reduced in HIF-1α-GFAP Cre+ mice as indicated by a decrease in proinflammatory cytokines and a decrease in the percentage of Gr1(hi) macrophages. Collectively, these studies have identified a novel function for HSCs and HIF-1α in orchestrating the clearance of necrotic cells from the liver and demonstrated a key role for HSCs in modulating macrophage phenotype during acute liver injury.

A parent-of-origin effect on honeybee worker ovary size.

Apis mellifera capensis is unique among honeybees in that unmated workers can produce pseudo-clonal female offspring via thelytokous parthenogenesis. Workers use this ability to compete among themselves and with their queen to be the mother of new queens. Males could therefore enhance their reproductive success by imprinting genes that enhance fertility in their daughter workers. This possibility sets the scene for intragenomic conflict between queens and drones over worker reproductive traits. Here, we show a strong parent-of-origin effect for ovary size (number of ovarioles) in reciprocal crosses between two honeybee subspecies, A. m. capensis and Apis mellifera scutellata. In this cross, workers with an A. m. capensis father had 30% more ovarioles than genotypically matched workers with an A. m. scutellata father. Other traits we measured (worker weight at emergence and the presence/absence of a spermatheca) are influenced more by rearing conditions than by parent-of-origin effects. Our study is the first to show a strong epigenetic (or, less likely, cytoplasmic maternal) effect for a reproductive trait in the honeybee and suggests that a search for parent-of-origin effects in other social insects may be fruitful.

Exposure of Ldlr-/- Mice to a PFAS Mixture and Outcomes Related to Circulating Lipids, Bile Acid Excretion, and the Intestinal Transporter ASBT.

BACKGROUND: Previous epidemiological studies have repeatedly found per- and polyfluoroalkyl substances (PFAS) exposure associated with higher circulating cholesterol, one of the greatest risk factors for development of coronary artery disease. The main route of cholesterol catabolism is through its conversion to bile acids, which circulate between the liver and ileum via enterohepatic circulation. Patients with coronary artery disease have decreased bile acid excretion, indicating that PFAS-induced impacts on enterohepatic circulation may play a critical role in cardiovascular risk. OBJECTIVES: Using a mouse model with high levels of low-density and very low-density lipoprotein (LDL and VLDL, respectively) cholesterol and aortic lesion development similar to humans, the present study investigated mechanisms linking exposure to a PFAS mixture with increased cholesterol. METHODS: Male and female Ldlr-/- mice were fed an atherogenic diet (Clinton/Cybulsky low fat, 0.15% cholesterol) and exposed to a mixture of 5 PFAS representing legacy, replacement, and emerging subtypes (i.e., PFOA, PFOS, PFHxS, PFNA, GenX), each at a concentration of 2mg/L, for 7 wk. Blood was collected longitudinally for cholesterol measurements, and mass spectrometry was used to measure circulating and fecal bile acids. Transcriptomic analysis of ileal samples was performed via RNA sequencing. RESULTS: After 7 wk of PFAS exposure, average circulating PFAS levels were measured at 21.6, 20.1, 31.2, 23.5, and 1.5µg/mL in PFAS-exposed females and 12.9, 9.7, 23, 14.3, and 1.7µg/mL in PFAS-exposed males for PFOA, PFOS, PFHxS, PFNA, and GenX, respectively. Total circulating cholesterol levels were higher in PFAS-exposed mice after 7 wk (352mg/dL vs. 415mg/dL in female mice and 392mg/dL vs. 488mg/dL in male mice exposed to vehicle or PFAS, respectively). Total circulating bile acid levels were higher in PFAS-exposed mice (2,978 pg/µL vs. 8,496 pg/µL in female mice and 1,960 pg/µL vs. 4,452 pg/µL in male mice exposed to vehicle or PFAS, respectively). In addition, total fecal bile acid levels were lower in PFAS-exposed mice (1,797 ng/mg vs. 682 ng/mg in females and 1,622 ng/mg vs. 670 ng/mg in males exposed to vehicle or PFAS, respectively). In the ileum, expression levels of the apical sodium-dependent bile acid transporter (ASBT) were higher in PFAS-exposed mice. DISCUSSION: Mice exposed to a PFAS mixture displayed higher circulating cholesterol and bile acids perhaps due to impacts on enterohepatic circulation. This study implicates PFAS-mediated effects at the site of the ileum as a possible critical mediator of increased cardiovascular risk following PFAS exposure. https://doi.org/10.1289/EHP14339.

Mixtures of per- and polyfluoroalkyl substances (PFAS) alter sperm methylation and long-term reprogramming of offspring liver and fat transcriptome.

Male fertility has been declining worldwide especially in countries with high levels of endocrine disrupting chemicals (EDCs). Per- and polyfluorinated alkyl Substances (PFAS) have been classified as EDCs and have been linked to adverse male reproductive health. The mechanisms of these associations and their implications on offspring health remain unknown. The aims of the current study were to assess the effect of PFAS mixtures on the sperm methylome and transcriptional changes in offspring metabolic tissues (i.e., liver and fat). C57BL/6 male mice were exposed to a mixture of PFAS (PFOS, PFOA, PFNA, PFHxS, Genx; 20 µg/L each) for 18-weeks or water as a control. Genome-wide methylation was assessed on F0 epidydimal sperm using reduced representation bisulfite sequencing (RRBS) and Illumina mouse methylation array, while gene expression was assessed by bulk RNA sequencing in 8-week-old offspring derived from unexposed females. PFAS mixtures resulted in 2,861 (RRBS) and 83 (Illumina) sperm DMRs (q < 0.05). Functional enrichment revealed that PFAS-induced sperm DMRs were associated with behavior and developmental pathways in RRBS, while Illumina DMRs were related to lipid metabolism and cell signaling. Additionally, PFAS mixtures resulted in 40 and 53 differentially expressed genes (DEGs) in the liver and fat of males, and 9 and 31 DEGs in females, respectively. Functional enrichment of DEGs revealed alterations in cholesterol metabolism and mitotic cell cycle regulation in the liver and myeloid leukocyte migration in fat of male offspring, while in female offspring, erythrocyte development and carbohydrate catabolism were affected in fat. Our results demonstrate that exposure to a mixture of legacy and newly emerging PFAS chemicals in adult male mice result in aberrant sperm methylation and altered gene expression of offspring liver and fat in a sex-specific manner. These data indicate that preconception PFAS exposure in males can be transmitted to affect phenotype in the next generation.

Interleukin-10 disrupts liver repair in acetaminophen-induced acute liver failure.

Introduction: Systemic levels of the anti-inflammatory cytokine interleukin 10 (IL-10) are highest in acetaminophen (APAP)-induced acute liver failure (ALF) patients with the poorest prognosis. The mechanistic basis for this counterintuitive finding is not known, as induction of IL-10 is hypothesized to temper the pathological effects of immune cell activation. Aberrant production of IL-10 after severe liver injury could conceivably interfere with the beneficial, pro-reparative actions of immune cells, such as monocytes. Methods: To test this possibility, we determined whether IL-10 levels are dysregulated in mice with APAP-induced ALF and further evaluated whether aberrant production of IL-10 prevents monocyte recruitment and/or the resolution of necrotic lesions by these cells. Results: Our studies demonstrate that in mice challenged with 300 mg/kg acetaminophen (APAP), a hepatotoxic dose of APAP that fails to produce ALF (i.e., APAP-induced acute liver injury; AALI), Ly6Chi monocytes were recruited to the liver and infiltrated the necrotic lesions by 48 hours coincident with the clearance of dead cell debris. At 72 hours, IL-10 was upregulated, culminating in the resolution of hepatic inflammation. By contrast, in mice treated with 600 mg/kg APAP, a dose that produces clinical features of ALF (i.e., APAP-induced ALF; AALF), IL-10 levels were markedly elevated by 24 hours. Early induction of IL-10 was associated with a reduction in the hepatic numbers of Ly6Chi monocytes resulting in the persistence of dead cell debris. Inhibition of IL-10 in AALF mice, beginning at 24 hours after APAP treatment, increased the hepatic numbers of monocytes which coincided with a reduction in the necrotic area. Moreover, pharmacologic elevation of systemic IL-10 levels in AALI mice reduced hepatic myeloid cell numbers and increased the area of necrosis. Discussion: Collectively, these results indicate that during ALF, aberrant production of IL-10 disrupts the hepatic recruitment of monocytes, which prevents the clearance of dead cell debris. These are the first studies to document a mechanistic basis for the link between high IL-10 levels and poor outcome in patients with ALF.

Exposure to per- and polyfluoroalkyl substances (PFAS) and type 2 diabetes risk.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous man-made chemicals found in consumer products including fabrics, food packaging, non-stick coatings, and aqueous film-forming foams. PFAS are stable and extremely resistant to degradation, resulting in high persistence throughout the environment as well as in human blood. PFAS consist of a large family of synthetic chemicals, with over 4000 distinct varieties having been identified and around 250 currently being manufactured at globally relevant levels. Numerous epidemiological studies have linked exposure to PFAS with adverse health effects ranging from immunotoxicity, cardiometabolic disease, developmental and reproductive effects, cancer, and recently type 2 diabetes. Several studies have demonstrated associations between serum PFAS concentrations and glycemic indicators of type 2 diabetes including glucose, insulin, and HOMA-IR in adolescent and adult cohorts. In addition, some studies have shown positive associations with incident type 2 diabetes and multiple PFAS. However, the link between PFAS exposure and the development of diabetes continues to be a disputed area of study, with conflicting data having been reported from various epidemiological studies. In this mini review we will summarize the current state of the literature linking PFAS to type 2 diabetes and discuss important future directions including the use of more complex mixtures-based statistical analyses.