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A method was developed to extract and quantify microcystins (MCs) from mouse liver with limits of quantification (LOQs) lower than previously reported. MCs were extracted from 40-mg liver samples using 85:15 (v:v) CH3CN:H2O containing 200 mM ZnSO4 and 1% formic acid. Solid-phase extraction with a C18 cartridge was used for sample cleanup. MCs were detected and quantified using HPLC-orbitrap-MS with simultaneous MS/MS detection of the 135.08 m/z fragment from the conserved Adda amino acid for structural confirmation. The method was used to extract six MCs (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, and MC-LW) from spiked liver tissue and the MC-LR cysteine adduct (MC-LR-Cys) created by the glutathione detoxification pathway. Matrix-matched internal standard calibration curves were constructed for each MC (R2 &ge, 0.993), with LOQs between 0.25 ng per g of liver tissue (ng/g) and 0.75 ng/g for MC-LR, MC-RR, MC-YR, MC-LA, and MC-LR-Cys, and 2.5 ng/g for MC-LF and MC-LW. The protocol was applied to extract and quantify MC-LR and MC-LR-Cys from the liver of mice that had been gavaged with 50 &micro, g or 100 &micro, g of MC-LR per kg bodyweight and were euthanized 2 h, 4 h, or 48 h after final gavage. C57Bl/6J (wild type, control) and Leprdb/J (experiment) mice were used as a model to study non-alcoholic fatty liver disease. The Leprdb/J mice were relatively inefficient in metabolizing MC-LR into MC-LR-Cys, which is an important defense mechanism against MC-LR exposure. Trends were also observed as a function of MC-LR gavage amount and time between final MC-LR gavage and euthanasia/organ harvest.

Background Epidemiological studies have shown Per- and polyfluoroalkyl substances (PFAS) to be associated with diseases of dysregulated lipid and sterol homeostasis such as steatosis and cardiometabolic disorders. However, the majority of mechanistic studies rely on single chemical exposures instead of identifying mechanisms related to the toxicity of PFAS mixtures. Objectives The goal of the current study is to investigate mechanisms linking exposure to a PFAS mixture with alterations in lipid metabolism, including increased circulating cholesterol and bile acids. Methods Male and female wild-type C57BL/6J mice were fed an atherogenic diet used in previous studies of pollutant-accelerated atherosclerosis and exposed to water containing a mixture of 5 PFAS representing legacy, replacement, and alternative subtypes (i.e., PFOA, PFOS, PFNA, PFHxS, and GenX), each at a concentration of 2 mg/L, for 12 weeks. Changes at the transcriptome and metabolome level were determined by RNA-seq and high-resolution mass spectrometry, respectively. Results We observed increased circulating cholesterol, sterol metabolites, and bile acids due to PFAS exposure, with some sexual dimorphic effects. PFAS exposure increased hepatic injury, demonstrated by increased liver weight, hepatic inflammation, and plasma alanine aminotransferase levels. Females displayed increased lobular and portal inflammation compared to the male PFAS-exposed mice. Hepatic transcriptomics analysis revealed PFAS exposure modulated multiple metabolic pathways, including those related to sterols, bile acids, and acyl carnitines, with multiple sex-specific differences observed. Finally, we show that hepatic and circulating levels of PFOA were increased in exposed females compared to males, but this sexual dimorphism was not the same for other PFAS examined. Discussion Exposure of mice to a mixture of PFAS results in PFAS-mediated modulation of cholesterol levels, possibly through disruption of enterohepatic circulation.

Brain-derived neurotrophic factor (BDNF) modulates the synaptic transmission of several monoaminergic neuronal systems, including forebrain dopamine-containing neurons. Recent evidence shows a strong correlation between neuropsychiatric disorders and BDNF hypofunction. The aim of the present study was to characterize the effect of low endogenous levels of BDNF on dopamine system function in the caudate-putamen using heterozygous BDNF (BDNF(+/-) ) mice. Apparent extracellular dopamine levels in the caudate-putamen, determined by quantitative microdialysis, were significantly elevated in BDNF(+/-) mice compared with wildtype controls (12 vs. 5 nM, respectively). BDNF(+/-) mice also had a potentiated increase in dopamine levels following potassium (120 mM)-stimulation (10-fold) relative to wildtype controls (6-fold). Slice fast-scan cyclic voltammetry revealed that BDNF(+/-) mice had reductions in both electrically evoked dopamine release and dopamine uptake rates in the caudate-putamen. Superfusion of BDNF led to partial recovery of the electrically stimulated dopamine release response in BDNF(+/-) mice. Conversely, tissue accumulation of L-3,4-dihydroxyphenylalanine, extracellular levels of dopamine metabolites, and spontaneous locomotor activity were unaltered. Together, this study indicates that endogenous BDNF influences dopamine system homeostasis by regulating the release and uptake dynamics of pre-synaptic dopamine transmission.

The risk of developing neurologic diseases and disorders differs between men and women. For example, men are approximately 1.5 times more likely than women to develop Parkinson’s disease, which is characterized by dopamine (DA) neuron degeneration in the striatum.1 Furthermore, women are twice as likely to develop depressive disorders compared to men.2 To better understand sex differences in these neurologic conditions, research has focused on the molecules implicated in these conditions, such as the neurotransmitters DA and serotonin, as well as the neurotrophic factors involved in the growth, differentiation, plasticity, and protection of neurons, like brain-derived neurotrophic factor (BDNF).3−5 To better understand sex differences in disease risk and progression, gonadal hormones such as estrogen and testosterone have also been investigated. In ovariectomized rats, a physiologic dose of estrogen increases striatal extracellular DA concentrations and tyrosine hydroxylase activity.6 Furthermore, estrogen stimulates similar downstream second messengers, ERK1 and ERK2, in a manner analogous to BDNF in cortical explant cultures.7 Additionally, female rats show increased depression vulnerability relative to their male counterparts when their BDNF levels are at their lowest, which correlates with their maximum estrogen levels.8−10 Sex differences are also observed in neurologic disorders such as addiction, with women and men responding differently to drugs of abuse. For example, in clinical studies, women are more likely to start using illicit drugs sooner and in greater quantities than men,11,12 whereas men are more likely to die from an overdose than women.13 In animal models, males are more susceptible to the neurotoxic effects of methamphetamine (METH) than females, as shown by reduced striatal DA and DA transporter (DAT) binding.14−16 These observations in animal and clinical studies, along with others using METH and incorporating estrogen and testosterone, have suggested that females may be protected from the harmful effects of drugs because of their naturally higher of estrogen levels.15,17,18 Numerous studies have elucidated the importance of BDNF in maintaining the function and survival of striatal DA neurons.19−22 To better understand the endogenous role of BDNF throughout the brain and body, we used genetically modified heterozygous (BDNF+/–) mice developed by Ernfors and colleagues.23 Male BDNF+/– mice have increased extracellular and intracellular DA levels compared to their male wildtype littermates,21,24,25 which are not associated with alterations in DAT. The objective of this study was to determine if these striatal DA alterations are present in female BDNF+/– mice. Using the complementary techniques of in vivo microdialysis and slice fast scan cyclic voltammetry (FSCV), we investigated DA dynamics in female wildtype and BDNF+/– mice.