Subcellular spatio-temporal intravital kinetics of aflatoxin B1 and ochratoxin A in liver and kidney

Abstract

Local accumulation of xenobiotics in human and animal tissues may cause adverse effects. Large differences in their concentrations may exist between individual cell types, often due to the expression of specific uptake and export carriers. Here we established a two-photon microscopy-based technique for spatio-temporal detection of the distribution of mycotoxins in intact kidneys and livers of anesthetized mice with subcellular resolution. The mycotoxins ochratoxin A (OTA, 10 mg/kg b.w.) and aflatoxin B1 (AFB1, 1.5 mg/kg b.w.), which both show blue auto-fluorescence, were analyzed after intravenous bolus injections. Within seconds after administration, OTA was filtered by glomeruli, and enriched in distal tubular epithelial cells (dTEC). A striking feature of AFB1 toxicokinetics was its very rapid uptake from sinusoidal blood into hepatocytes (t1/2 ~ 4 min) and excretion into bile canaliculi. Interestingly, AFB1 was enriched in the nuclei of hepatocytes with zonal differences in clearance. In the cytoplasm of pericentral hepatocytes, the half-life (t1/2~ 63 min) was much longer compared to periportal hepatocytes of the same lobules (t1/2 ~ 9 min). In addition, nuclear AFB1 from periportal hepatocytes cleared faster compared to the pericentral region. These local differences in AFB1 clearance may be due to the pericentral expression of cytochrome P450 enzymes that activate AFB1 to protein- and DNA-binding metabolites. In conclusion, the present study shows that large spatio-temporal concentration differences exist within the same tissues and its analysis may provide valuable additional information to conventional toxicokinetic studies.

Spatio-Temporal Multiscale Analysis of Western Diet-Fed Mice Reveals a Translationally Relevant Sequence of Events during NAFLD Progression

“…Intravital imaging of lipid droplets and macrophages in mouse livers was performed using an inverted two-photon microscope LSM MP7 (Zeiss, Germany), as previously described [19][20][21]. Briefly, after induction of anesthesia in mice [21], bolus tail vein injections of Hoechst 33258 (nucleus marker), TMRE/Rhodamine123 (markers of lobular zonation and mitochondrial membrane potential), Bodipy (lipid marker), and/or F4/80 antibody (macrophage marker) were performed approximately 30 min before the start of recording (Table 4). The time point 9 weeks was selected to represent the early stage (3-12 weeks) after WD feeding as the lipid droplet zonation was clearly visible.…”

Hypoalbuminemia affects the spatio-temporal tissue distribution of ochratoxin A in liver and kidneys: consequences for organ toxicity

“…Intravital imaging of OTA transport in the livers and the kidneys of anesthetized wild-type and albumin knockout mice was done using an inverted two-photon microscope (LSM MP7, Zeiss, Germany). Details of the surgical preparation of the mice and exposure of the liver and the kidneys for imaging were previously described (Ghallab et al 2021a ; Reif et al 2017 ). Tissue morphology was visualized by intravenous administration of the mitochondrial membrane potential marker tetramethylrhodamine ethyl ester (TMRE) (#T669, ThermoFisher Scientific) approximately 10 min before recording (Table 2 ).…”

“…These analyses can be performed in intact livers of anesthetized mice (Remetic et al 2022 ). Moreover, compound filtration from renal capillaries into Bowman’s space, and the further transport into the lumen of tubules and subsequent uptake into the cytoplasm of tubular epithelial cells can be analyzed by this technique (Ghallab et al 2021a ; Koeppert et al 2021 ; Reif et al 2017 ).…”

“…Ochratoxin A (OTA), a mycotoxin of relevance for human health (EFSA 2020 ), has a high plasma protein-binding capacity (> 99%); it is actively taken up from the sinusoidal blood into hepatocytes by organic anion-transporting polypeptides (OATPs), and then actively exported from hepatocytes into bile canaliculi by multidrug resistance-associated protein 2 (MRP2 or ABCC2) and breast cancer resistance protein (BCRP) (Table 1 ) (Anzai et al 2010 ; Ringot et al 2006 ). Based on its auto-fluorescence properties, label-free intravital imaging of OTA has already been established (Ghallab et al 2021a ), which permits us to re-evaluate two already-published concepts: (1) “If a drug is highly bound to plasma proteins and has a high extraction ratio by the liver, which involves active transport mechanisms to concentrate the drug in the hepatocytes, then plasma proteins act as delivery systems” (Fanali et al 2012 ), and (2) “If the protein-bound drug cannot readily leave the capillaries, only the unbound drug can be distributed to tissues, therefore having pharmacological activity, as well as toxic effects” (Fanali et al 2012 ). Following up of these two concepts may lead to contradicting conclusions.…”

Colchicine overdose impairs the capacity of Kupffer cells to clear foreign particles and endotoxins

“…To visualize bile canaliculi, a bolus of the bile acid analogue cholyl-lysyl-fluorescein (CLF; #451041, Corning) was intravenously injected via a tail vein catheter approximately 15 min before the end of the recording (Vartak et al 2021 ). Image analysis was done in defined regions of interest using Zen software (Zeiss, Germany), as previously described (Ghallab et al 2021a ; Koeppert et al 2021 ). Representative video/image of at least 4 mice per condition was analyzed and presented in the result section.…”