MultiFlow ® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/β-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition.PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.
K E Y W O R D SDNA damage, flow cytometry, metabolic activation, γH2AX, p53
Emerging studies have highlighted the disproportionate role of
Candida albicans
in influencing both early community assembly of the bacterial microbiome and dysbiosis during allergic diseases and intestinal inflammation. Nonpathogenic colonization of the human gastrointestinal (GI) tract by
C. albicans
is common, and the role of this single fungal species in modulating bacterial community reassembly after broad-spectrum antibiotics can be readily recapitulated in mouse studies.
Noradrenaline (norepinephrine) is known to modulate many physiological functions and behaviors. In this study, we tested to what extent astrocytes, a type of glial cell, participate in noradrenergic signaling in mouse primary visual cortex (V1). Astrocytes are essential partners of neurons in the central nervous system. They are central to brain homeostasis, but also dynamically regulate neuronal activity, notably by relaying and regulating neuromodulator signaling. Indeed, astrocytes express receptors for multiple neuromodulators, including noradrenaline, but the extent to which astrocytes are involved in noradrenergic signaling remains unclear. To test whether astrocytes are involved in noradrenergic neuromodulation in mice, we knocked down the major noradrenaline receptor in astrocytes, the alpha 1A-adrenoreceptor. Using this model, we found that the alpha 1A-adrenoreceptor is involved in triggering intracellular calcium transients in astrocytes, which are generally thought to underlie astrocyte function. To test if impaired alpha 1A-adrenoreceptor signaling in astrocytes affected the function of neuronal circuits in V1, we used electrophysiological measurements and found that noradrenergic signaling through astrocyte alpha 1A-adrenoreceptor controls the basal level of inhibition and regulates plasticity in V1 by potentiating synaptic responses in circuits involved in visual information processing.
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