Characterization of ochratoxin A transport by human organic anion transporters

doi:10.1016/S0024-3205(01)01296-6

Life Sciences

Volume 69, Issue 18, 21 September 2001, Pages 2123-2135

https://doi.org/10.1016/S0024-3205(01)01296-6 Get rights and content

Abstract

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporters (hOAT1 and hOAT3, respectively) using the second segment of proximal tubule (S₂) cells from mice stably expressing hOAT1 and hOAT3 (S₂ hOAT1 and S₂ hOAT3). S₂ hOAT1 and S₂ hOAT3 exhibited a time- and dose-dependent, and a saturable increase in uptake of [³H]-OTA, with apparent K_m values of 0.42 μM (hOAT1) and 0.75 μM (hOAT3). These OTA uptakes were inhibited by several substrates for the OATs. Para-aminohippuric acid (PAH), probenecid, piroxicam, octanoate and citrinin inhibited [³H]-OTA uptake by hOAT1 and hOAT3 in a competitive manner (K_i = 4.29∼3080 μM), with the following order of potency: probenecid > octanoate > PAH > piroxicam > citrinin for hOAT1; probenecid > piroxicam > octanoate> citrinin > PAH for hOAT3. These results indicate that hOAT1, as well as hOAT3, mediates a high-affinity transport of OTA on the basolateral side of the proximal tubule, but hOAT1- and hOAT3-mediated OTA transport are differently influenced by the substrates for the OATs. These pharmacological characteristics of hOAT1 and hOAT3 may be significantly related with the events in the development of OTA-induced nephrotoxicity in the human kidney.

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Cited by (93)

Investigating the interaction between organic anion transporter 1 and ochratoxin A: An in silico structural study to depict early molecular events of substrate recruitment and the impact of single point mutations
2022, Toxicology Letters
Organic anion transporters (OATs) belong to a subgroup of the solute carrier 22 transporter family. OATs have a central role in xenobiotic disposition affecting the toxicokinetics of its substrates and inter-individual differences in their expression, activity and function impact both toxicokinetics and toxicodynamics. Amongst OATs, OAT1 (solute carrier family 22 member 6) is involved in the urinary excretion of many xenobiotics bringing substrates into renal proximal tubular cells which can then be secreted across the apical membrane into the tubule lumen. The mycotoxin ochratoxin A has been shown to have a high affinity for OAT1, which is an important renal transporter involved in its urinary excretion. Nowadays, molecular modeling techniques are widely applied to assess protein-ligand interactions and may provide a tool to depict the mechanic of xenobiotic action be it toxicokinetics or toxicodynamics. This work provides a structured pipeline consisting of docking and molecular dynamic simulations to study OAT1-ligand interactions and the impact of OAT1 polymorphisms on such interactions. Such a computational structure-based analytical framework allowed to: i) model OAT1-substrate complex formation and depict the features correlating its sequence, structure and its capability to recruit substrates; and ii) investigate the impact of OAT1 missense mutations on substrate recruitment. Perspectives on applying such a structured pipeline to xenobiotic-metabolising enzymes are discussed.
A key role for the transporter OAT1 in systemic lipid metabolism
2021, Journal of Biological Chemistry
Organic anion transporter 1 (OAT1/SLC22A6) is a drug transporter with numerous xenobiotic and endogenous substrates. The Remote Sensing and Signaling Theory suggests that drug transporters with compatible ligand preferences can play a role in “organ crosstalk,” mediating overall organismal communication. Other drug transporters are well known to transport lipids, but surprisingly little is known about the role of OAT1 in lipid metabolism. To explore this subject, we constructed a genome-scale metabolic model using omics data from the Oat1 knockout mouse. The model implicated OAT1 in the regulation of many classes of lipids, including fatty acids, bile acids, and prostaglandins. Accordingly, serum metabolomics of Oat1 knockout mice revealed increased polyunsaturated fatty acids, diacylglycerols, and long-chain fatty acids and decreased ceramides and bile acids when compared with wildtype controls. Some aged knockout mice also displayed increased lipid droplets in the liver when compared with wildtype mice. Chemoinformatics and machine learning analyses of these altered lipids defined molecular properties that form the structural basis for lipid-transporter interactions, including the number of rings, positive charge/volume, and complexity of the lipids. Finally, we obtained targeted serum metabolomics data after short-term treatment of rodents with the OAT-inhibiting drug probenecid to identify potential drug–metabolite interactions. The treatment resulted in alterations in eicosanoids and fatty acids, further supporting our metabolic reconstruction predictions. Consistent with the Remote Sensing and Signaling Theory, the data support a role of OAT1 in systemic lipid metabolism.
Microphysiological system modeling of ochratoxin A-associated nephrotoxicity
2020, Toxicology
Citation Excerpt :
This suggests a critical role of probenecid-sensitive active transporter(s) in OTA uptake from the basolateral aspect into kidney epithelial cells, presumably attributable to organic anion transporter(s) (OAT) 1 and/or 3. This is in accordance with the report that OAT transports OTA using a gene-overexpression systems (Anzai et al., 2010; Jung et al., 2001). Since OATs are highly expressed in the kidney, the presence of an active uptake process by OATs may contribute to kidney-specific exposure of OTA, leading to a subsequent increase in intracellular concentration of OTQ/OTHQ, which results in more severe kidney toxicity compared with other organs.
Ochratoxin A (OTA) is one of the most abundant mycotoxin contaminants in food stuffs and possesses carcinogenic, nephrotoxic, teratogenic, and immunotoxic properties. Specifically, a major concern is severe nephrotoxicity, which is characterized by degeneration of epithelial cells of the proximal tubules and interstitial fibrosis. However, the mechanism of OTA toxicity, as well as the genetic risk factors contributing to its toxicity in humans has been elusive due to the lack of adequate models that fully recapitulate human kidney function in vitro. The present study attempts to evaluate dose-response relationships, identify the contribution of active transport proteins that govern the renal disposition of OTA, and determine the role of metabolism in the bioactivation and detoxification of OTA using a 3D human kidney proximal tubule microphysiological system (kidney MPS). We demonstrated that LC₅₀ values of OTA in kidney MPS culture (0.375–1.21 μM) were in agreement with clinically relevant toxic concentrations of OTA in urine. Surprisingly, no enhancement of kidney injury biomarkers was evident in the effluent of the kidney MPS after OTA exposure despite significant toxicity observed by LIVE/DEAD staining. Instead, these biomarkers decreased in an OTA concentration-dependent manner. Furthermore, the effect of 1-aminobenzotriazole (ABT) and 6-(7-Nitro-2,1,3-benzoxadiazol-4-ylthio) hexanol (NBDHEX), pan-inhibitors of P450 and glutathione S-transferase (GST) enzymes, respectively, on OTA-induced toxicity in kidney MPS was examined. These studies revealed significant enhancement of OTA-induced toxicity by NBDHEX (3 μM) treatment, whereas ABT (1 mM) treatment decreased OTA-induced toxicity, suggesting roles for GSTs and P450 enzymes in the detoxification and bioactivation of OTA, respectively. Analysis of transcriptional changes using RNA-sequencing of kidney MPS treated with different concentrations of OTA revealed downregulation of several nuclear factor (erythroid derived-2)-like 2 (NRF2)-regulated genes by OTA treatment, including GSTs. The transcriptional repression of GSTs is likely playing a key role in OTA toxicity via attenuation of glutathione conjugation/detoxification. The sequential molecular events may explain the mechanism of toxicity associated with OTA. Additionally, OTA transport studies using kidney MPS in the presence and absence of probenecid (1 mM) suggested a role for organic anionic membrane transporter(s) in the kidney specific disposition of OTA. Our findings provide a clearer understanding of the mechanism of OTA-induced kidney injury, which may support changes in risk assessment, regulatory agency policies on allowable exposure levels, and determination of the role of genetic factors in populations at risk for OTA nephrotoxicity.
Mitigation of cell apoptosis induced by ochratoxin A (OTA) is possibly through organic cation transport 2 (OCT2) knockout
2018, Food and Chemical Toxicology
Citation Excerpt :
OTA induced apparent kidney and DNA damage but caused a finite amount of oxidative stress after the rats were exposed to OTA for 13 weeks (Qi et al., 2014). Previous research suggests that OAT1 and OAT3 may be involved in the transport of OTA (Tsuda et al., 1999; Jung et al., 2001). In our study, the OCT2 knockout cell model and OCT2 overexpressing cell model were constructed to explore the relationship between OCT2 and apoptosis, as well as the role of OTA-induced cell toxicity.
Ochratoxin A (OTA) is a secondary metabolite of fungi such as Aspergillus ochraceus, A. niger and A. carbonarius, Penicillium verrucosum, and various other Penicillium, Petromyces, and Neopetromyces species. Various foods can be contaminated with OTA, potentially causing several toxic effects such as nephrotoxicity, hepatotoxicity and neurotoxicity. Typically, OTA is excreted by organic anion transporters (OATs). There is no research indicating organic cation transporters (OCTs) are involved in OTA nephrotoxicity.
In our study, NRK-52E cells and rats were treated with OTA. OTA changed the expression of OCT1, OCT2 and OCT3 in NRK-52E cells and rat kidneys. TEA alleviated OTA-induced cell death, apoptosis, and DNA damage, and increased ROS. The OCT2 knockout cell line was constructed by the CRISPR/Cas 9 system. OCT2 knockout did not change the gene expression of OCT1, OAT1 and OAT3. OCT2 knockout alleviated the increase of Caspase 3 and CDK1 induced by OTA, leading to a reduction of apoptosis. In addition, OCT2 overexpression increased cell toxicity and expression of Caspase 3. In short, our findings indicate that OCT2 knockout possibly mitigate OTA-induced apoptosis by preventing the increase of Caspase 3 and CDK1.
Uptake Transporters
2018, Comprehensive Toxicology: Third Edition
Membrane transporters are recognized to be an important class of proteins for regulating cellular and physiologic solute and fluid balance. Transporter proteins can be generally separated into two major classes—uptake and efflux transporters. Not only are transporter proteins critical to human physiology in the maintenance of normal homeostasis via transport of endogenous substrates, but they are also, in many cases, important to the disposition of numerous xenobiotic compounds such as environmental toxins, dietary constituents, drugs, and their metabolites. In particular, over the past two decades, members of the solute carrier (SLC) families, including the organic anion transporting polypeptide (SLCO) family, the organic cation/anion/zwitterion transporter (SLC22) family, and the sodium bile salt cotransport (SLC10) family, have been determined to have important physiological, pathological, and therapeutic implications. In this article, we focus on uptake transporter proteins and provide a comprehensive overview of the significant roles these proteins play in normal physiology and the drug disposition process.
Ochratoxin A transport by the human breast cancer resistance protein (BCRP), multidrug resistance protein 2 (MRP2), and organic anion-transporting polypeptides 1A2, 1B1 and 2B1
2017, Toxicology and Applied Pharmacology
Ochratoxin A (OTA) is a fungal secondary metabolite that can contaminate various foods. OTA has several toxic effects like nephrotoxicity, hepatotoxicity, and neurotoxicity in different animal species, but its mechanisms of toxicity are still unclear. How OTA accumulates in kidney, liver, and brain is as yet unknown, but transmembrane transport proteins are likely involved. We studied transport of OTA in vitro, using polarized MDCKII cells transduced with cDNAs of the efflux transporters mouse (m)Bcrp, human (h)BCRP, mMrp2, or hMRP2, and HEK293 cells overexpressing cDNAs of the human uptake transporters OATP1A2, OATP1B1, OATP1B3, or OATP2B1 at pH 7.4 and 6.4. MDCKII-mBcrp cells were more resistant to OTA toxicity than MDCKII parental and hBCRP-transduced cells. Transepithelial transport experiments showed some apically directed transport by MDCKII-mBcrp cells at pH 7.4, whereas both mBcrp and hBCRP clearly transported OTA at pH 6.4. There was modest transport of OTA by mMrp2 and hMRP2 only at pH 6.4. OATP1A2 and OATP2B1 mediated uptake of OTA both at pH 7.4 and 6.4, but OATP1B1 only at pH 7.4. There was no detectable transport of OTA by OATP1B3. Our data indicate that human BCRP and MRP2 can mediate elimination of OTA from cells, thus reducing OTA toxicity. On the other hand, human OATP1A2, OATP1B1, and OATP2B1 can mediate cellular uptake of OTA, which could aggravate OTA toxicity.

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Original articlesCharacterization of ochratoxin A transport by human organic anion transporters

Abstract

Original articles
Characterization of ochratoxin A transport by human organic anion transporters