Elemental Composition of Commercially Available Cannabis Rolling Papers.

With the recent legalization of cannabis in multiple jurisdictions and widespread use as a medical treatment, there has been an increased focus on product safety and the potential impacts of contaminants on human health. One factor that has received little attention is the possible exposure to potentially hazardous levels of toxic elements from rolling (smoking) papers. The elemental composition of rolling papers is largely unregulated, with a minority of jurisdictions regulating papers only when they are part of a final cannabis product. This study reports the concentrations of 26 elements in commercially available rolling papers and estimates potential maximum exposures relative to USP232 and ICH Q3D dosages in pharmaceutical compounds. Exposure estimates indicate that the concentrations of several elements in some products, particularly Cu, Cr, and V, may present a potential hazard to frequent users. Several elements, including Ag, Ca, Ba, Cu, Ti, Cr, Sb, and possibly others, are likely present in elevated quantities in some papers due to product design and manufacturing processes. Our results further suggest that Cu-based pigments are used by a number of manufacturers and that regular use of these products might result in exposures as high as 4.5-11 times the maximum exposure limits. Further research to quantify the contribution of rolling papers to elemental exposure under realistic smoking conditions is warranted.

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.

Identification of Serine-Containing Microcystins by UHPLC-MS/MS Using Thiol and Sulfoxide Derivatizations and Detection of Novel Neutral Losses.

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria, and their structural diversity has led to the discovery of more than 300 congeners to date. However, with known amino acid combinations, many more MC congeners are theoretically possible, suggesting many remain unidentified. Herein, two novel serine (Ser)-containing MCs were putatively identified in a Lake Erie cyanobacterial harmful algal bloom (cyanoHAB), using high-resolution UHPLC-MS as well as thiol and sulfoxide derivatization procedures. These MCs contain an α,ß-unsaturated carbonyl on methyl dehydroalanine (Mdha) residue that undergoes Michael addition to produce a thiol-derivatized MC. Derivatization reactions using various thiolation reagents were followed by MS/MS, and two Python codes were used for data analysis and structural elucidation of MCs. Two novel MCs containing Ser at position 1 (i.e., next to Mdha) were putatively identified as [Ser1]MC-RR and [Ser1]MC-YR. Using thiol- and sulfoxide-modified [Ser1]MCs, identifications were confirmed by the observation of specific neutral losses of the oxidized thiols or sulfoxides in CID-MS/MS spectra in both positive and negative electrospray ionization (ESI) modes. These novel neutral losses are unique for MCs with Mdha and an adjacent Ser residue. Data suggest that a gas-phase reaction occurs between oxygen from adjacent Ser residue and sulfur of the Mdha-bonded thiol or sulfoxide, which leads to the formation and detection of stable cyclic MC ions in MS/MS spectra at m/z values corresponding to the loss of oxidized thiols or oxidized sulfoxides from Ser1-containing MCs.

An assessment of a biosensor system for the quantification of microcystins in freshwater cyanobacterial blooms.

Microcystin-producing cyanobacterial blooms are a global issue threatening drinking water supplies and recreation on lakes and beaches. Direct measurement of microcystins is the only way to ensure waters have concentrations below guideline concentrations; however, analyzing water for microcystins takes several hours to days to obtain data. We tested LightDeck Diagnostics' bead beater cell lysis and two versions of the quantification system designed to give microcystin concentrations within 20 min and compared it to the standard freeze-thaw cycle lysis method and ELISA quantification. The bead beater lyser was only 30 % effective at extracting microcystins compared to freeze-thaw. When considering freeze-thaw samples analyzed in 2021, there was good agreement between ELISA and LightDeck version 2 (n = 152; R2 = 0.868), but the LightDeck slightly underestimated microcystins (slope of 0.862). However, we found poor relationships between LightDeck version 2 and ELISA in 2022 (n = 49, slopes 0.60 to 1.6; R2 < 0.6) and LightDeck version 1 (slope = 1.77 but also a high number of less than quantifiable concentrations). After the quantification issues are resolved, combining the LightDeck system with an already-proven rapid lysis method (such as microwaving) will allow beach managers and water treatment operators to make quicker, well-informed decisions.

Aerosolized Cyanobacterial Harmful Algal Bloom Toxins: Microcystin Congeners Quantified in the Atmosphere.

Cyanobacterial harmful algal blooms (cHABs) have the potential to adversely affect public health through the production of toxins such as microcystins, which consist of numerous molecularly distinct congeners. Microcystins have been observed in the atmosphere after emission from freshwater lakes, but little is known about the health effects of inhaling microcystins and the factors contributing to microcystin aerosolization. This study quantified total microcystin concentrations in water and aerosol samples collected around Grand Lake St. Marys (GLSM), Ohio. Microcystin concentrations in water samples collected on the same day ranged from 13 to 23 µg/L, dominated by the d-Asp3-MC-RR congener. In particulate matter <2.5 µm (PM2.5), microcystin concentrations up to 156 pg/m3 were detected; the microcystins were composed primarily of d-Asp3-MC-RR, with additional congeners (d-Asp3-MC-HtyR and d-Asp3-MC-LR) observed in a sample collected prior to a storm event. The PM size fraction containing the highest aerosolized MC concentration ranged from 0.44 to 2.5 µm. Analysis of total bacteria by qPCR targeting 16S rDNA revealed concentrations up to 9.4 × 104 gc/m3 in aerosol samples (≤3 µm), while a marker specific to cyanobacteria was not detected in any aerosol samples. Concentrations of aerosolized microcystins varied even when concentrations in water were relatively constant, demonstrating the importance of meteorological conditions (wind speed and direction) and aerosol generation mechanism(s) (wave breaking, spillway, and aeration systems) when evaluating inhalation exposure to microcystins and subsequent impacts on human health.

Elucidating microbial mechanisms of microcystin-LR degradation in Lake Erie beach sand through metabolomics and metatranscriptomics.

As one of five Laurentian Great Lakes, Lake Erie ranks among the top freshwater drinking sources and ecosystems globally. Historical and current agriculture mismanagement and climate change sustains the environmental landscape for late summer cyanobacterial harmful algal blooms, and consequently, cyanotoxins such as microcystin (MC). Microcystin microbial degradation is a promising mitigation strategy, however the mechanisms controlling the breakdown of MCs in Lake Erie are not well understood. Pelee Island, Ontario, Canada is located in the western basin of Lake Erie and the bacterial community in the sand has demonstrated the capacity of metabolizing the toxin. Through a multi-omic approach, the metabolic, functional and taxonomical signatures of the Pelee Island microbial community during MC-LR degradation was investigated over a 48-hour period to comprehensively study the degradation mechanism. Cleavage of bonds surrounding nitrogen atoms and the upregulation of nitrogen deamination (dadA, alanine dehydrogenase, leucine dehydrogenase) and assimilation genes (glnA, gltB) suggests a targeted isolation of nitrogen by the microbial community for energy production. Methylotrophic pathways RuMP and H4MPT control assimilation and dissimilation of carbon, respectively and differential abundance of Methylophilales indicates an interconnected role through electron exchange of denitrification and methylotrophic pathways. The detected metabolites did not resolve a clear breakdown pathway, but rather the diversity of products in combination with taxonomic and functional results supports that a variety of strategies are applied, such as epoxidation, hydroxylation, and aromatic degradation. Annual repeated exposure to the toxin may have allowed the community to adaptatively establish a novel pathway through functional plasticity and horizontal gene transfer. The culmination of these results reveals the complexity of the Pelee Island sand community and supports a dynamic and cooperative metabolism between microbial species to achieve MC degradation.

Microcystin congeners in Lake Erie follow the seasonal pattern of nitrogen availability.

Cyanobacteria harmful algal blooms produce many toxic secondary metabolites called cyanotoxins. The most studied group of cyanotoxins are microcystins (MC), with over 300 congeners reported. MC-LR is the most studied congener because of its abundance and toxicity. Recent toxicology studies suggest that more hydrophobic MC congeners such as MC-LA, MC-LF, and MC-LW may be less abundant but up to seven times more toxic than MC-LR, whereas, MC-RR's toxicity is only one-fifth that of MC-LR. Hence, understanding the environmental stressors that change the MC congener profile is critical to assessing the negative impact on environmental and human health. A two-year field and experimental study investigated seasonal and spatial changes of MC congener profiles in the western basin of Lake Erie. Both studies showed that nitrogen enrichment favored the production of nitrogen-rich MC-RR (C49H75N13O12). The field study showed that nitrogen depletion favored the low-nitrogen MC-LA (C46H67N7O12). MC-LR (a medium N level, C49H75N10O12) accounted for ∼30% to 50% of the total MC concentration and was stable across nitrogen concentrations. Using the relative toxicity and concentrations of each MC congener, both LC-MS/MS and ELISA overestimated the toxicity early bloom (July) and underestimated it late bloom (September). On 24 July 2019, highly toxic MC-LW and MC-LF were detected at nearshore stations with relative toxicity exceeding drinking water standards. This study demonstrated that the less toxic, high nitrogen MC-RR dominated under nitrogen-replete conditions in the early season, whereas the more toxic, less nitrogen MC-LA dominated under nitrogen-limited conditions later in the season.

Intraspecific genetic variation is critical to robust toxicological predictions of aquatic contaminants.

Environmental risk assessment is a critical tool for protecting aquatic life and its effectiveness is predicated on predicting how natural populations respond to contaminants. Yet, routine toxicity testing typically examines only one genotype, which may render risk assessments inaccurate as populations are most often composed of genetically distinct individuals. To determine the importance of intraspecific variation in the translation of toxicity testing to populations, we quantified the magnitude of genetic variation within 20 Daphnia magna clones derived from one lake using whole genome sequencing and phenotypic assays. We repeated these assays across two exposure levels of microcystins, a cosmopolitan and lethal aquatic contaminant produced by harmful algal blooms. We found considerable intraspecific genetic variation in survival, growth, and reproduction, which was amplified by microcystins exposure. Finally, using simulations we demonstrate that the common practice of employing a single genotype to calculate toxicity tolerance failed to produce an estimate within the 95% confidence interval over half of the time. These results illuminate the importance of incorporating intraspecific genetic variation into toxicity testing to reliably predict how natural populations will respond to aquatic contaminants.

Microcystin concentrations and congener composition in relation to environmental variables across 440 north-temperate and boreal lakes.

Understanding the environmental conditions and taxa that promote the occurrence of cyanobacterial toxins is imperative for effective management of lake ecosystems. Herein, we modeled total microcystin presence and concentrations with a broad suite of environmental predictors and cyanobacteria community data collected across 440 Canadian lakes using standardized methods. We also conducted a focused analysis targeting 14 microcystin congeners across 190 lakes, to examine how abiotic and biotic factors influence their relative proportions. Microcystins were detected in 30 % of lakes, with the highest total concentrations occurring in the most eutrophic lakes located in ecozones of central Canada. The two most commonly detected congeners were MC-LR (61 % of lakes) and MC-LA (37 % of lakes), while 11 others were detected more sporadically across waterbodies. Congener diversity peaked in central Canada where cyanobacteria biomass was highest. Using a zero-altered hurdle model, the probability of detecting microcystin was best explained by increasing Microcystis biomass, Daphnia and cyclopoid biomass, soluble reactive phosphorus, pH and wind. Microcystin concentrations increased with the biomass of Microcystis and other less dominant cyanobacteria taxa, as well as total phosphorus, cyclopoid copepod biomass, dissolved inorganic carbon and water temperature. Collectively, these models accounted for 34 % and 70 % of the variability, respectively. Based on a multiple factor analysis of microcystin congeners, cyanobacteria community data, environmental and zooplankton data, we found that the relative abundance of most congeners varied according to trophic state and were related to a combination of cyanobacteria genera biomasses and environmental variables.

Adipogenic and endocrine disrupting mixture effects of organic and inorganic pollutant mixtures.

Chronic health conditions are rapidly increasing in prevalence and cost to society worldwide: in the US, >42 % of adults aged 20 and older are currently classified as obese. Exposure to endocrine disrupting chemicals (EDCs) has been implicated as a causal factor; some EDCs, termed "obesogens", can increase weight and lipid accumulation and/or perturb metabolic homeostasis. This project aimed to assess the potential combination effects of diverse inorganic and organic contaminant mixtures, which more closely reflect environmentally realistic exposures, on nuclear receptor activation/inhibition and adipocyte differentiation. Herein, we focused on two polychlorinated biphenyls (PCB-77 and 153), two perfluoroalkyl substances (PFOA and PFOS), two brominated flame retardants (PBB-153 and BDE-47), and three inorganic contaminants (lead, arsenic, and cadmium). We examined adipogenesis using human mesenchymal stem cells and receptor bioactivities using luciferase reporter gene assays in human cell lines. We observed significantly greater effects for several receptor bioactivities by various contaminant mixtures relative to individual components. All nine contaminants promoted triglyceride accumulation and/or pre-adipocyte proliferation in human mesenchymal stem cells. Comparing simple component mixtures to individual components at 10 % and 50 % effect levels revealed putative synergistic effects for each of the mixtures for at least one of the concentrations relative to the individual component chemicals, some of which also exhibited significantly greater effects than the component contaminants. Our results support further testing of more realistic and complex contaminant mixtures that better reflect environmental exposures, in order to more conclusively define mixture responses both in vitro and in vivo.

Deep Cyanobacteria Layers: An Overlooked Aspect of Managing Risks of Cyanobacteria.

The risk of human exposure to cyanotoxins is partially influenced by the location of toxin-producing cyanobacteria in waterbodies. Cyanotoxin production can occur throughout the water column, with deep water production representing a potential public health concern, specifically for drinking water supplies. Deep cyanobacteria layers are often unreported, and it remains to be seen if lower incident rates reflect an uncommon phenomenon or a monitoring bias. Here, we examine Sunfish Lake, Ontario, Canada as a case study lake with a known deep cyanobacteria layer. Cyanotoxin and other bioactive metabolite screening revealed that the deep cyanobacteria layer was toxigenic [0.03 µg L-1 microcystins (max) and 2.5 µg L-1 anabaenopeptins (max)]. The deep layer was predominantly composed of Planktothrix isothrix (exhibiting a lower cyanotoxin cell quota), with Planktothrix rubescens (exhibiting a higher cyanotoxin cell quota) found at background levels. The co-occurrence of multiple toxigenic Planktothrix species underscores the importance of routine surveillance for prompt identification leading to early intervention. For instance, microcystin concentrations in Sunfish Lake are currently below national drinking water thresholds, but shifting environmental conditions (e.g., in response to climate change or nutrient modification) could fashion an environment favoring P. rubescens, creating a scenario of greater cyanotoxin production. Future work should monitor the entire water column to help build predictive capacities for identifying waterbodies at elevated risk of developing deep cyanobacteria layers to safeguard drinking water supplies.

Quantification of microcystin production and biodegradation rates in the western basin of Lake Erie.

Cyanobacterial biomass forecasts currently cannot predict the concentrations of microcystin, one of the most ubiquitous cyanotoxins that threaten human and wildlife health globally. Mechanistic insights into how microcystin production and biodegradation by heterotrophic bacteria change spatially and throughout the bloom season can aid in toxin concentration forecasts. We quantified microcystin production and biodegradation during two growth seasons in two western Lake Erie sites with different physicochemical properties commonly plagued by summer Microcystis blooms. Microcystin production rates were greater with elevated nutrients than under ambient conditions and were highest nearshore during the initial phases of the bloom, and production rates were lower in later bloom phases. We examined biodegradation rates of the most common and toxic microcystin by adding extracellular stable isotope-labeled microcystin-LR (1 µg L-1), which remained stable in the abiotic treatment (without bacteria) with minimal adsorption onto sediment, but strongly decreased in all unaltered biotic treatments, suggesting biodegradation. Greatest biodegradation rates (highest of -8.76 d-1, equivalent to the removal of 99.98% in 18 h) were observed during peak bloom conditions, while lower rates were observed with lower cyanobacteria biomass. Cell-specific nitrogen incorporation from microcystin-LR by nanoscale imaging mass spectrometry showed that a small percentage of the heterotrophic bacterial community actively degraded microcystin-LR. Microcystin production and biodegradation rates, combined with the microcystin incorporation by single cells, suggest that microcystin predictive models could be improved by incorporating toxin production and biodegradation rates, which are influenced by cyanobacterial bloom stage (early vs. late bloom), nutrient availability, and bacterial community composition.

Lead exposure induces nitrative stress and disrupts ribbon synapses in the cochlea.

Environmental exposure to heavy metal lead, a public health hazard in many post-industrial cities, causes hearing impairment upon long-term exposure. Lead-induced cochlear and vestibular dysfunction is well-documented in animal models. Although short-term exposure to lead at concentrations relevant to environmental settings does not cause significant shifts in hearing thresholds in adults, moderate- to low-level lead exposures induce neuronal damage and synaptic dysfunction. We reported that lead exposure induces oxidative stress in the mouse cochlea. However, lead-induced nitrative stress and potential damage to cochlear ribbon synapses are yet to be fully understood. Therefore, this study has evaluated cochlear synaptopathy and nitrative stress in young-adult mice exposed to 2 mM lead acetate for 28 days. Inductively coupled plasma mass spectrometry (ICP-MS) analysis indicated that this exposure significantly increased the blood lead levels. Assessment of hair cell loss by immunohistochemistry analysis and outer hair cell (OHC) activity by recording distortion product otoacoustic emissions (DPOAEs) indicated that the structure and function of the hair cells were not affected by lead exposure. However, this exposure significantly decreased the expression of C-terminal-binding protein-2 (CtBP2) and GluA2, pre- and post-synaptic protein markers in the inner hair cell synapses, particularly in the basal turn of the organ of Corti, suggesting lead-induced disruption of ribbon synapses. In addition, lead exposure significantly increased the nitrotyrosine levels in spiral ganglion cells, suggesting lead-induced nitrative stress in the cochlea. Collectively, these findings suggest that lead exposure even at levels that do not affect the OHCs induces cochlear nitrative stress and causes cochlear synaptopathy.

As We Drink and Breathe: Adverse Health Effects of Microcystins and Other Harmful Algal Bloom Toxins in the Liver, Gut, Lungs and Beyond.

Freshwater harmful algal blooms (HABs) are increasing in number and severity worldwide. These HABs are chiefly composed of one or more species of cyanobacteria, also known as blue-green algae, such as Microcystis and Anabaena. Numerous HAB cyanobacterial species produce toxins (e.g., microcystin and anatoxin-collectively referred to as HAB toxins) that disrupt ecosystems, impact water and air quality, and deter recreation because they are harmful to both human and animal health. Exposure to these toxins can occur through ingestion, inhalation, or skin contact. Acute health effects of HAB toxins have been well documented and include symptoms such as nausea, vomiting, abdominal pain and diarrhea, headache, fever, and skin rashes. While these adverse effects typically increase with amount, duration, and frequency of exposure, susceptibility to HAB toxins may also be increased by the presence of comorbidities. The emerging science on potential long-term or chronic effects of HAB toxins with a particular emphasis on microcystins, especially in vulnerable populations such as those with pre-existing liver or gastrointestinal disease, is summarized herein. This review suggests additional research is needed to define at-risk populations who may be helped by preventative measures. Furthermore, studies are required to develop a mechanistic understanding of chronic, low-dose exposure to HAB toxins so that appropriate preventative, diagnostic, and therapeutic strategies can be created in a targeted fashion.

Identification of Novel Microcystins Using High-Resolution MS and MSn with Python Code.

Cyanotoxins called microcystins (MCs) are highly toxic and can be present in drinking water sources. Determining the structure of MCs is paramount because of its effect on toxicity. Though over 300 MC congeners have been discovered, many remain unidentified. Herein, a method is described for the putative identification of MCs using liquid chromatography (LC) coupled with high-resolution (HR) Orbitrap mass spectrometry (MS) and a new bottom-up sequencing strategy. Maumee River water samples were collected during a harmful algal bloom and analyzed by LC-MS with simultaneous HRMS and MS/MS. Unidentified ions with characteristic MC fragments (135 and 213 m/z) were recognized as possible novel MC congeners. An innovative workflow was developed for the putative identification of these ions. Python code was written to generate the potential structures of unidentified MCs and to assign ions after the fragmentation for structural confirmation. The workflow enabled the putative identification of eight previously reported MCs for which standards are not available and two newly discovered congeners, MC-HarR and MC-E(OMe)R.

Toward Revealing Microcystin Distribution in Mouse Liver Tissue Using MALDI-MS Imaging.

Cyanotoxins can be found in water and air during cyanobacterial harmful algal blooms (cHABs) in lakes and rivers. Therefore, it is very important to monitor their potential uptake by animals and humans as well as their health effects and distribution in affected organs. Herein, the distribution of hepatotoxic peptide microcystin-LR (MC-LR) is investigated in liver tissues of mice gavaged with this most common MC congener. Preliminary matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging experiments performed using a non-automated MALDI matrix deposition device and a MALDI-time-of-flight (TOF) mass spectrometer yielded ambiguous results in terms of MC-LR distribution in liver samples obtained from MC-LR-gavaged mice. The tissue preparation for MALDI-MS imaging was improved by using an automated sprayer for matrix deposition, and liver sections were imaged using an Nd:YAG MALDI laser coupled to a 15 Tesla Fourier-transform ion cyclotron resonance (FT-ICR)-mass spectrometer. MALDI-FT-ICR-MS imaging provided unambiguous detection of protonated MC-LR (calculated m/z 995.5560, z = +1) and the sodium adduct of MC-LR (m/z 1017.5380, z = +1) in liver sections from gavaged mice with great mass accuracy and ultra-high mass resolution. Since both covalently bound and free MC-LR can be found in liver of mice exposed to this toxin, the present results indicate that the distribution of free microcystins in tissue sections from affected organs, such as liver, can be monitored with high-resolution MALDI-MS imaging.

The Lake Erie HABs Grab: A binational collaboration to characterize the western basin cyanobacterial harmful algal blooms at an unprecedented high-resolution spatial scale.

Monitoring of cyanobacterial bloom biomass in large lakes at high resolution is made possible by remote sensing. However, monitoring cyanobacterial toxins is only feasible with grab samples, which, with only sporadic sampling, results in uncertainties in the spatial distribution of toxins. To address this issue, we conducted two intensive "HABs Grabs" of microcystin (MC)-producing Microcystis blooms in the western basin of Lake Erie. These were one-day sampling events during August of 2018 and 2019 in which 100 and 172 grab samples were collected, respectively, within a six-hour window covering up to 2,270 km2 and analyzed using consistent methods to estimate the total mass of MC. The samples were analyzed for 57 parameters, including toxins, nutrients, chlorophyll, and genomics. There were an estimated 11,513 kg and 30,691 kg of MCs in the western basin during the 2018 and 2019 HABs Grabs, respectively. The bloom boundary poses substantial issues for spatial assessments because MC concentration varied by nearly two orders of magnitude over very short distances. The MC to chlorophyll ratio (MC:chl) varied by a factor up to 5.3 throughout the basin, which creates challenges for using MC:chl to predict MC concentrations. Many of the biomass metrics strongly correlated (r > 0.70) with each other except chlorophyll fluorescence and phycocyanin concentration. While MC and chlorophyll correlated well with total phosphorus and nitrogen concentrations, MC:chl correlated with dissolved inorganic nitrogen. More frequent MC data collection can overcome these issues, and models need to account for the MC:chl spatial heterogeneity when forecasting MCs.

Exposure to a mixture of legacy, alternative, and replacement per- and polyfluoroalkyl substances (PFAS) results in sex-dependent modulation of cholesterol metabolism and liver injury.

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.

Pesticide exposure and adverse health effects associated with farmwork in Northern Thailand.

OBJECTIVES: To assess pesticide exposure and understand the resultant health effects of agricultural workers in Northern Thailand. METHODS: This was a cross-sectional study. We quantified exposure to pesticides, including chlorpyrifos, methomyl, and metalaxyl, by air sampling and liquid chromatography/mass spectrometry. We estimated differences in self-reported health outcomes, complete blood counts, cholinesterase activity, and serum/urine calcium and creatinine concentrations at baseline between farmworkers and comparison workers, and after pesticide spraying in farmworkers only. RESULTS: This study included 97 men between the ages of 22 and 76 years; 70 were conventional farmworkers; and 27 did not report any prior farmwork or pesticide spraying. None of the farmworkers wore standardized personal protective equipment (PPE) for the concentrated chemicals they were working with. Methomyl (8.4-13 481.9 ng/m3 ), ethyl chlorpyrifos (11.6-67 759 ng/m3 ), and metalaxyl (13.9-41 191.3 ng/m3 ) were detected via personal air sampling. When it came to reporting confidence in the ability to handle personal problems, only 43% of farmworkers reported feeling confident, which reflects higher stress levels in comparison to 78% of comparison workers (P = .028). Farmworkers also had significantly lower monocyte counts (P = .01), serum calcium (P = .01), red blood count (P = .01), white blood cell count (P = .04), and butyrylcholinesterase activity (P < .0001), relative to comparison workers. After adjusting for body mass index (BMI), age, and smoking, methomyl air concentrations were associated with a decrease in farmworker acetylcholinesterase activity (beta = -0.327, P = .016). CONCLUSIONS: This population of farmworkers had significant alterations in stress measures and clinical biomarkers, including decreased blood cell counts and cholinesterase activity, relative to matched controls. These changes are potentially linked to occupational pesticide exposures. Improving PPE use presents a likely route for preventive intervention in this population.

Negative Regulation of Human Hepatic Constitutive Androstane Receptor by Cholesterol Synthesis Inhibition: Role of Sterol Regulatory Element Binding Proteins.

The squalene synthase inhibitor squalestatin 1 (Squal1) is a potent and efficacious inducer of CYP2B expression in primary cultured rat hepatocytes and rat liver. To determine whether Squal1 is also an inducer of human CYP2B, the effects of Squal1 treatment were evaluated in primary cultured human hepatocytes, differentiated HepaRG cells, and humanized mouse livers. Squal1 treatment did not increase CYP2B6 mRNA levels in human hepatocytes or HepaRG cells and only slightly and inconsistently increased CYP2B6 mRNA content in humanized mouse liver. However, treatment with farnesol, which mediates Squal1's effect on rat CYP2B expression, increased CYP2B6 mRNA levels in HepaRG cells expressing the constitutive androstane receptor (CAR), but not in cells with knocked-down CAR. To determine the impact of cholesterol biosynthesis inhibition on CAR activation, the effects of pravastatin (Prava) were determined on CITCO-mediated gene expression in primary cultured human hepatocytes. Prava treatment abolished CITCO-inducible CYP2B6 expression, but had less effect on rifampicin-mediated CYP3A4 induction, and CITCO treatment did not affect Prava-inducible HMG-CoA reductase (HMGCR) expression. Treatment with inhibitors of different steps of cholesterol biosynthesis attenuated CITCO-mediated CYP2B6 induction in HepaRG cells, and Prava treatment increased HMGCR expression and inhibited CYP2B6 induction with comparable potency. Transfection of HepG2 cells with transcriptionally active sterol regulatory element binding proteins (SREBPs) reduced CAR-mediated transactivation, and inducible expression of transcriptionally active SREBP2 attenuated CITCO-inducible CYP2B6 expression in HepaRG cells. These findings suggest that Squal1 does not induce CYP2B6 in human hepatocytes because Squal1's inhibitory effect on cholesterol biosynthesis interferes with CAR activation. SIGNIFICANCE STATEMENT: The cholesterol biosynthesis inhibitor squalestatin 1 induces rat hepatic CYP2B expression indirectly by causing accumulation of an endogenous isoprenoid that activates the constitutive androstane receptor (CAR). This study demonstrates that squalestatin 1 does not similarly induce CYP2B6 expression in human hepatocytes. Rather, inhibition of cholesterol biosynthesis interferes with CAR activity, likely by activating sterol regulatory element binding proteins. These findings increase our understanding of the endogenous processes that modulate human drug-metabolizing gene expression.