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Exosomes are secreted membrane-bound vesicles containing a cargo of curated nucleic acids, proteins, and lipids that can alter gene expression in recipient cells. Toxic agents can alter exosome synthesis and bioactive cargo composition, thus allowing exosomes to serve as biomarkers of exposure and response. While human and animal studies have identified exosome biomarkers of organ toxicity, in vitro models are ideal to examine biological mechanisms of exosome function. Here, we discuss the importance of exosomes in toxicology research and describe applications of in vitro models in advancing our understanding of their role in exposure-associated disease. This discussion of new research frontiers is in commemoration of the invaluable contributions of Dr. Daniel Acosta to the field of in vitro biology and toxicology. Emerging studies have implicated exosomes as mediators of neurodegeneration by shuttling pollutant-induced pathogenic proteins and miRNAs from afflicted neurons to neighboring cells. Exosomes also provide a mechanistic link between inhalation exposures and airway inflammation, remodeling, and systemic effects. Exosomes provide the means for toxic agents to initiate oncogenic transformation and create favorable tumor microenvironments. Furthermore, exosome-mediated drug delivery can alter drug pharmacologic profiles. Expansion in this field using in vitro models is essential to unlock the potential applications of exosome biology in toxicology., Competing Interests: Conflict of interest The authors declare no conflict of interest. In addition, Drs. Bowers and Ramos filed a patent “Exosome-based Liquid Biopsy” pending., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Multiple in vitro model systems are currently available to evaluate structure and function relationships in the cardiovascular system as well as the system's response to injury. As the level of molecular sophistication continues to advance, so does the level of complexity of the analysis. One of the most daunting tasks faced by researchers interested in studying cardiovascular function and injury is the selection of the system or systems best suited to answer the particular question at hand. In order to successfully apply any given model system, the researcher must recognize the advantages and limitations in the system of choice. This review provides a listing of the historical and modern techniques used to study cardiovascular function and chemically-induced toxicity. With the growing number of new pharmaceuticals discovered each year, it is imperative to use experimental model systems that allow for identification of targets that participate in or mediate adverse outcomes. Clearly, in vitro analysis cannot replace in vivo experimentation, but the methods currently available allow for a reduction in the number of animals used for experimentation and a better understanding of the complexity associated with the injury response.

In the present study, the time- and concentration-dependent effects of the carcinogen benzo[a]pyrene (BaP) on c-fos, c-jun, c-myc and c-Ha-ras expression were evaluated in primary cultured rat hepatocytes. These genes exhibited differential patterns of expression with peak mRNA levels at different times following incubation in the presence of serum mitogens. The expression of c-fos and c-jun was markedly inhibited by BaP (3 mum), with pronounced effects observed during the early part of the induction response. In contrast, the kinetics of c-myc and c-Ha-ras inducibility were enhanced by BaP (3 mum) leading to significant enhancement of mRNA levels. The gene modulatory effects of BaP were concentration dependent in the range of 0.3-30 mum. Challenge with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (1 nm), an aryl hydrocarbon receptor (AhR) agonist, elicited responses comparable to BaP for c-jun and c-Ha-ras, but not for c-fos or c-myc. alpha-naphthoflavone (10 nm), an antagonist/agonist of the AhR and inhibitor of cytochrome P450, elicited c-jun and c-Ha-ras responses comparable to BaP. Pretreatment of the cells with this agent nullified the modulatory effects of BaP on c-fos and c-myc. These results suggest that deregulation of growth-related gene expression in cultured rat hepatocytes by BaP is mediated by complex mechanisms that may involve AhR signalling and oxidative metabolism of the parent compound.

The cytotoxic responses of cultured rat renal cortico-tubular epithelial cells (TECs) and glomerular mesangial cells (GMCs) to selected polycyclic and polyhalogenated aromatic hydrocarbons (AHs) were evaluated to further define class specific differences in toxicity potential among these chemicals. Primary cultures of renal cells were exposed to benzo[a]pyrene (BaP, 0.3-30mum); naphthalene (NAPH, 1-1000mum); 2-methylnaphthalene (2-MNAPH, 1-1000mum), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 0.01-1nm); 2,3,7,8-tetrachlorodibenzofuran (TCDF, 0.05-5nm; and pentachlorophenol (PCP, 0.1-10mum for 4 or 24hr. Measurements of mitochondrial membrane fragility in TECs revealed that the polycyclic aromatic hydrocarbons (PAHs) (BaP, NAPH and 2-MNAPH) preferentially injured these cells relative to halogenated aromatic hydrocarbons (HAHs). Extended exposures to all AHs tested were associated with moderate mitochondrial injury in TECs. Challenge of TECs with AHs for 4hr did not modulate GSH levels, although modest increases in this tripeptide occurred on extended exposures. Similar response profiles were observed in GMCs, where PAHs elicited mitochondrial damage by 4hr, while extended challenge was associated with injury in response to all AHs. Only aryl hydrocarbon receptor (AhR) ligands (BaP, TCDD and TCDF) depleted intracellular glutathione (GSH) in GMCs, while extended exposures to BaP and TCDD, as well as NAPH and 2-MNAPH, were associated with rebound increases in cellular GSH content. These data indicate that AHs can compromise renal cell function by interference with mitochondrial function and GSH homeostasis and implicate both epithelial and mesenchymal populations in the nephrotoxic response to this heterogeneous class of chemicals.

Cultured rat hepatocytes were challenged with benzo[a]pyrene (BaP; 0.3-30 mum), naphthalene (NAPH; 0.1-100 mum), 2-methylnaphthalene (2-MNAPH; 0.1-100 mum), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; 0.001-1 nM), 2,3,7,8-tetrachlorodibenzofuran (TCDF; 0.005-5 nM) or pentachlorophenol (PCP; 0.1-100 mum) for 4-24 hr to define class-specific differences in hepatotoxic potential. Mitochondrial fragility and GSH status were monitored as indices of hepatocyte injury. A 4-hr challenge with BaP and PCP increased mitochondrial fragility in a concentration-dependent manner, while TCDD and TCDF elicited erratic increases, and NAPH and 2-MNAPH were without effect. Pretreatment of hepatocytes with 250 muM diamide enhanced the mitochondrial toxicity of BaP. The aryl hydrocarbon receptor agonists upregulated hepatocyte GSH levels by 24 hr, a response which in the case of BaP was preceded by varying degrees of GSH depletion between 6 to 16 hr. NAPH and 2-MNAPH transiently decreased hepatocyte GSH levels at 12 hr, but were without effect at later time points, while PCP did not modulate hepatocyte GSH levels. Modulation of hepatocyte GSH by BaP and TCDD was antagonized by 10 muM alpha-naphthoflavone. These data implicate oxidative mechanisms and aryl hydrocarbon receptor signalling in aromatic hydrocarbon hepatotoxicity.

The present studies were conducted to assess cell-specific cytotoxic responses of the kidney to selected aromatic hydrocarbons (AHs) using in vitro culture systems of glomerular mesangial cells and cortical tubular epithelial cells. Primary cultures of glomerular mesangial cells or cortical tubular epithelial cells were treated for 24 hr with naphthalene (NAPH, 0.1-1000 mum), 2-methylnaphthalene (2-MNAPH, 0.1-1000 mum), benzo[a]pyrene (B[a]P, 0.3-300 mum), pentachlorophenol (PCP, 0.1-1000 mum), 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 0.001-1 nm) or 2,3,7,8-tetrachlorodibenzofuran (TCDF, 0.005-5 nm). [(3)H]Thymidine incorporation into DNA, cellular potassium content, and lactate dehydrogenase (LDH) leakage were used as indices of cytotoxicity. Glomerular mesangial cells exposed to NAPH or 2-MNAPH (0.1-1000 mum) exhibited [(3)H]thymidine incorporation profiles similar to those of the vehicle controls, whereas B[a]P at all concentrations and PCP at 1000 mum decreased [(3)H]thymidine incorporation in comparison with the controls. TCDD and TCDF increased [(3)H]thymidine incorporation relative to controls at 0.1 nm and 0.5 nm, respectively. Glomerular mesangial cells treated with NAPH or PCP exhibited decreased cellular potassium content and increased LDH leakage only at the 1000 mum concentration. In tubular epithelial cells, treatment with NAPH (10 or 1000 mum), 2-MNAPH (10 or 1000 mum), B[a]P (300 mum), and PCP (10 or 1000 mum) induced concentration-dependent decreases in [(3)H]thymidine incorporation relative to controls. In contrast, exposure of tubular epithelial cells to TCDD or TCDF did not modulate [(3)H]thymidine incorporation at any of the concentrations tested. A modest decrease in cellular potassium content and an increase in LDH leakage was observed at the 1000 mum concentration of PCP in tubular epithelial cells. On the basis of these data, we conclude that renal glomerular mesangial cells and tubular epithelial cells exhibit differential cytotoxic response profiles to AHs.