Accumulation, Depuration, and Biological Effects of Polystyrene Microplastic Spheres and Adsorbed Cadmium and Benzo(a)pyrene on the Mussel Mytilus galloprovincialis

Filter feeders are target species for microplastic (MP) pollution, as particles can accumulate in the digestive system, disturbing feeding processes and becoming internalized in tissues. MPs may also carry pathogens or pollutants present in the environment. This work assessed the influence of polystyrene (PS) MP size and concentration on accumulation and depuration time and the role of MPs as vectors for metallic (Cd) and organic (benzo(a)pyrene, BaP) pollutants. One-day exposure to pristine MPs induced a concentration-dependent accumulation in the digestive gland (in the stomach and duct lumen), and after 3-day depuration, 45 µm MPs appeared between gill filaments, while 4.5 µm MPs also occurred within gill filaments. After 3-day exposure to contaminated 4.5 µm MPs, mussels showed increased BaP levels whilst Cd accumulation did not occur. Here, PS showed higher affinity to BaP than to Cd. Three-day exposure to pristine or contaminated MPs did not provoke significant alterations in antioxidant and peroxisomal enzyme activities in the gills and digestive gland nor in lysosomal membrane stability. Exposure to dissolved contaminants and to MP-BaP caused histological alterations in the digestive gland. In conclusion, these short-term studies suggest that MPs are ingested and internalized in a size-dependent manner and act as carriers of the persistent organic pollutant BaP.

Catalase deficiency facilitates the shuttling of free fatty acid to brown adipose tissue through lipolysis mediated by ROS during sustained fasting

Background Fatty acids (FA) derived from adipose tissue and liver serve as the main fuel in thermogenesis of brown adipose tissue (BAT). Catalase, a peroxisomal enzyme, plays an important role in maintaining intracellular redox homeostasis by decomposing hydrogen peroxide to either water or oxygen that oxidize and provide fuel for cellular metabolism. Although the antioxidant enzymatic activity of catalase is well known, its role in the metabolism and maintenance of energy homeostasis has not yet been revealed. The present study investigated the role of catalase in lipid metabolism and thermogenesis during nutrient deprivation in catalase-knockout (KO) mice. Results We found that hepatic triglyceride accumulation in KO mice decreased during sustained fasting due to lipolysis through reactive oxygen species (ROS) generation in adipocytes. Furthermore, the free FA released from lipolysis were shuttled to BAT through the activation of CD36 and catabolized by lipoprotein lipase in KO mice during sustained fasting. Although the exact mechanism for the activation of the FA receptor enzyme, CD36 in BAT is still unclear, we found that ROS generation in adipocytes mediated the shuttling of FA to BAT. Conclusions Taken together, our findings uncover the novel role of catalase in lipid metabolism and thermogenesis in BAT, which may be useful in understanding metabolic dysfunction. Graphical Abstract

Lifespan of Catalase-Deficient Mice Decreases Due to Lysosomal Dysfunction

Background: Lysosomes are a central hub for cellular metabolism and are involved in the regulation of cell homeostasis through the degradation or recycling of unwanted or dysfunctional organelles through the autophagy pathway. Catalase, a peroxisomal enzyme, plays an important role in cellular antioxidant defense by decomposing hydrogen peroxide into water and oxygen. In accordance with pleiotropic significance, both impaired lysosomes and catalase have been linked to many age-related pathologies with a decline in lifespan. Aging is characterized by progressive accumulation of macromolecular damage and the production of high levels of reactive oxygen species (ROS). Although lysosomes degrade the most long-lived proteins and organelles via the autophagic pathway, the role of lysosomes and their effect on peroxisomes during aging is not known. The present study investigated the role of catalase and lysosomal function in catalase-knockout (KO) mice.Results: We found that catalase-deficient mice exhibited an aging phenotype faster than wild-type (WT) mice. We also found that aged catalase-KO mice induced leaky lysosomes by progressive accumulation of lysosomal contents, such as cathespin D, into the cytosol. Leaky lysosomes inhibited autophagosome formation and triggered impaired autophagy. The dysregulation of autophagy triggered mTORC1 (mechanistic target of rapamycin complex 1) activation, which plays a pivotal role in modulating aging. However, the antioxidant N-acetyl-L-cysteine (NAC) and mTORC1 inhibitor rapamycin rescued leaky lysosomes and aging phenotypes in catalase-deficient aged mice.Conclusion: This study unveils the new role of catalase and its role in lysosomal function during aging.

Catalase deficiency facilitates the shuttling of free fatty acid to brown adipose tissue through lipolysis mediated by ROS during sustained fasting

Background Fatty acids (FA) derived from adipose tissue and liver serve as the main fuel in thermogenesis of brown adipose tissue (BAT). Catalase, a peroxisomal enzyme, plays an important role in maintaining intracellular redox homeostasis by decomposing hydrogen peroxide to either water or oxygen that oxidize and provide fuel for cellular metabolism. Although the antioxidant enzymatic activity of catalase is well known, its role in the metabolism and maintenance of energy homeostasis has not yet been revealed. The present study investigated the role of catalase in lipid metabolism and thermogenesis during nutrient deprivation in catalase-knockout (KO) mice. Results We found that hepatic triglyceride accumulation in KO mice decreased during sustained fasting due to lipolysis through reactive oxygen species (ROS) generation in adipocytes. Furthermore, the free FA released from lipolysis were shuttled to BAT through the activation of CD36 and catabolized by lipoprotein lipase in KO mice during sustained fasting. Although the exact mechanism for the activation of the FA receptor enzyme is still unclear, we found that ROS generation in adipocytes mediated the shuttling of FA to BAT. Conclusions Taken together, our findings uncover the novel role of catalase in lipid metabolism and thermogenesis in BAT, which may be useful in understanding metabolic dysfunction.

LC-MS Based Platform Simplifies Access to Metabolomics for Peroxisomal Disorders

Peroxisomes are central hubs for cell metabolism and their dysfunction is linked to devastating human disorders, such as peroxisomal biogenesis disorders and single peroxisomal enzyme/protein deficiencies. For decades, biochemical diagnostics have been carried out using classical markers such as very long-chain fatty acids (VLCFA), which can be inconspicuous in milder and atypical cases. Holistic metabolomics studies revealed several potentially new biomarkers for peroxisomal disorders for advanced laboratory diagnostics including atypical cases. However, establishing these new markers is a major challenge in routine diagnostic laboratories. We therefore investigated whether the commercially available AbsoluteIDQ p180 kit (Biocrates Lifesciences), which utilizes flow injection and liquid chromatography mass spectrometry, may be used to reproduce some key results from previous global metabolomics studies. We applied it to serum samples from patients with mutations in peroxisomal target genes PEX1, ABCD1, and the HSD17B4 gene. Here we found various changes in sphingomyelins and lysophosphatidylcholines. In conclusion, this kit can be used to carry out extended diagnostics for peroxisomal disorders in routine laboratories, even without access to a metabolomics unit.

The peroxisomal enzyme, FAR1, is induced during ER stress in an ATF6-dependent manner in cardiac myocytes

While peroxisomes have been extensively studied in other cell types, their presence and function have gone virtually unexamined in cardiac myocytes. Here, in neonatal rat ventricular myocytes (NRVM) we showed that several known peroxisomal proteins co-localize to punctate structures with a morphology typical of peroxisomes. Surprisingly, we found that the peroxisomal protein, fatty acyl-CoA reductase 1 (FAR1), was upregulated by chemical and pathophysiological ER stress induced by tunicamycin (TM) and simulated ischemia/reperfusion (sI/R), respectively. Moreover, FAR1 induction in NRVM was mediated by the ER stress-sensor, activating transcription factor 6 (ATF6). Functionally, FAR1 knockdown reduced myocyte death during oxidative stress induced by either sI/R or hydrogen peroxide (H2O2). Thus, Far1 is an ER stress-inducible gene, which encodes a protein that localizes to peroxisomes of cardiac myocytes, where it reduces myocyte viability during oxidative stress. Since FAR1 is critical for plasmalogen synthesis, these results imply that plasmalogens may exert maladaptive effects on the viability of myocytes exposed to oxidative stress.

ACOX2 is a prognostic marker and impedes the progression of hepatocellular carcinoma via PPARα pathway

Hepatocellular carcinoma (HCC) has been extensively studied as one of the most aggressive tumors worldwide. However, its mortality rate remains high due to ideal diagnosis and treatment strategies. Uncovering novel genes with prognostic significance would shed light on improving the HCC patient’s outcome. In our study, we applied data-independent acquisition (DIA) quantitative proteomics to investigate the expression landscape of 24 paired HCC patients. A total of 1029 differentially expressed proteins (DEPs) were screened. Then, we compared DEPs in our cohort with the differentially expressed genes (DEGs) in The Cancer Genome Atlas, and investigated their prognostic significance, and found 183 prognosis-related genes (PRGs). By conducting protein–protein interaction topological analysis, we identified four subnetworks with prognostic significance. Acyl-CoA oxidase 2 (ACOX2) is a novel gene in subnetwork1, encodes a peroxisomal enzyme, and its function in HCC was investigated in vivo and in vitro. The lower expression of ACOX2 was validated by real-time quantitative PCR, immunohistochemistry, and Western blot. Cell Counting Kit-8 assay, wound healing, and transwell migration assay were applied to evaluate the impact of ACOX2 overexpression on the proliferation and migration abilities in two liver cancer cell lines. ACOX2 overexpression, using a subcutaneous xenograft tumor model, indicated a tumor suppressor role in HCC. To uncover the underlying mechanism, gene set enrichment analysis was conducted, and peroxisome proliferator-activated receptor-α (PPARα) was proposed to be a potential target. In conclusion, we demonstrated a PRG ACOX2, and its overexpression reduced the proliferation and metastasis of liver cancer in vitro and in vivo through PPARα pathway.

Acquired deficiency of the peroxisomal enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase is a metabolic vulnerability in hepatoblastoma

Metabolic reprogramming provides transformed cells with proliferative and/or survival advantages. However, capitalizing on this therapeutically has been only moderately successful due to the relatively small magnitude of these differences and because cancers may re-program their metabolism to evade metabolic pathway inhibition. Mice lacking the peroxisomal bi-functional enzyme enoyl-CoA hydratase/3-hydroxyacyl CoA dehydrogenase (Ehhadh) and supplemented with the 12-carbon fatty acid lauric acid (C12) accumulate dodecanedioic acid (DDDA), a toxic C12 metabolite that causes hepatocyte necrosis and acute liver failure. In a murine model of pediatric hepatoblastoma (HB), down-regulation of Ehhadh also occurs in combination with a more general suppression of mitochondrial β- and peroxisomal ω-fatty acid oxidation (FAO) pathways. HB-bearing mice provided with C12 and/or DDDA-supplemented diets survived significantly longer than those on standard diets. The tumors also developed massive necrosis in response to short-term DDDA supplementation. Reduced Ehhadh was noted in murine hepatocellular carcinomas (HCCs) and in substantial subsets of human cancers, including HCCs. Acquired DDDA resistance was not associated with Ehhadh re-expression but was associated with 129 transcript differences ~90% of which were down-regulated in DDDA-resistant tumors and ~two-thirds of which correlated with survival in several human cancers. These transcripts often encoded components of the extracellular matrix suggesting that DDDA resistance arises from its reduced intracellular transport. Our results demonstrate the feasibility of a metabolic intervention that is non-toxic, inexpensive and likely compatible with traditional therapies. C12 and/or DDDA-containing diets could potentially be used to supplement other treatments or as alternative therapeutic choices.

Assessment of the Mode of Action Underlying the Effects of GenX in Mouse Liver and Implications for Assessing Human Health Risks

GenX is an alternative to environmentally persistent long-chain perfluoroalkyl and polyfluoroalkyl substances. Mice exposed to GenX exhibit liver hypertrophy, elevated peroxisomal enzyme activity, and other apical endpoints consistent with peroxisome proliferators. To investigate the potential role of peroxisome proliferator-activated receptor alpha (PPARα) activation in mice, and other molecular signals potentially related to observed liver changes, RNA sequencing was conducted on paraffin-embedded liver sections from a 90-day subchronic toxicity study of GenX conducted in mice. Differentially expressed genes were identified for each treatment group, and gene set enrichment analysis was conducted using gene sets that represent biological processes and known canonical pathways. Peroxisome signaling and fatty acid metabolism were among the most significantly enriched gene sets in both sexes at 0.5 and 5 mg/kg GenX; no pathways were enriched at 0.1 mg/kg. Gene sets specific to the PPARα subtype were significantly enriched. These findings were phenotypically anchored to histopathological changes in the same tissue blocks: hypertrophy, mitoses, and apoptosis. In vitro PPARα transactivation assays indicated that GenX activates mouse PPARα. These results indicate that the liver changes observed in GenX-treated mice occur via a mode of action (MOA) involving PPARα, an important finding for human health risk assessment as this MOA has limited relevance to humans.

Primary hyperoxaluria: diagnostics, treatment, outcomes

Primary hyperoxaluria (PH) is a rare autosomal recessive disease caused by defects in liver glyoxylate metabolism and leading to overproduction of oxalates. Of the three types of PH, type I is the most common and severe form of the disease, which is caused by deficiency or loss of the liver-specific, vitamin B6-dependent, peroxisomal enzyme alanine-glyoxylateaminotransferase (AGT). In all types of PH, urinary excretion of oxalate is strongly elevated (> 1 mmol /1,73 m2/24 h), which results in recurrent urolithiasis and/or progressive nephrocalcinosis and subsequently, with a decrease in glomerular filtration rate (GFR), to the deposition of oxalates in the tissues of the body and the development of systemic oxalosis. PH type I is diagnosed late, in > 30% of patients already at the terminal stage of renal disease (ESRD). Every fourth patient with PH type II achieves ESRD, but cases of ESRD in type III are extremely rare. The diagnosis of PH is based on clinical and imaging (ultrasound, X-ray, CT scan) findings, urine oxalate assessment, genetic analysis. Early initiation of conservative treatment (high fluid intake, sodium citrate, etc.) is aimed at preserving renal function. Pyridoxine treatment can be effective in about 30% of patients with PH type I. Time on dialysis in anticipation of transplantation should be short to avoid overt systemic oxalosis. Transplantation methods depend on the type of PH and on the degree of GFR reduction, but combined liver and kidney transplantation is the method of choice in patients with primary hyperoxaluria type I. High index of clinical suspicion of PH must be in patients with nephrocalcinosis and/or recurrent urolithiasis, especially if urinary stones are predominantly whewellite (calcium oxalate monohydrate) in order to start early conservative treatment and preserve kidney function.