Transport Of Ochratoxin A Research Articles

Characterization of Human Organic Anion Transporter 4 (hOAT4) and Mouse Oat5 (mOat5) As Functional Orthologs for Renal Anion Uptake and Efflux Transport.

Organic anions (OAs) are compounds including drugs or toxicants that are negatively charged at physiologic pH and are typically transported by organic anion transporters (OATs). Human OAT4 (SLC22A11) is expressed in the apical membrane of renal proximal tubules. Although there is no rodent ortholog of hOAT4, rodents express Oat5 (Slc22a19), an anion exchanger that is also localized to the apical membrane of renal proximal tubule cells. The purpose of this study was to determine the functional similarity between mouse Oat5 and human OAT4. Chinese hamster ovary (CHO) cells expressing SLC22A11 or Slc22a19 were used to assess the transport characteristics of radiolabeled ochratoxin (OTA). We determined the kinetics of OTA transport; the resulting Michaelis constant (Kt) and maximal rate of mediated substrate transport (Jmax) values were very similar for both hOAT4 and mOat5: Kt 3.9 and 7.2 μM, respectively, and Jmax 4.4 and 3.9 pmol/cm2, respectively. For the profile of OTA inhibition by OAs, IC50 values were determined for several clinically important drugs and toxicants. The resulting IC50 values ranged from 9 μM for indomethacin to ∼600 μM for the diuretic hydrochlorothiazide. We measured the efflux of OTA from preloaded cells; both hOAT4 and mOat5 supported the efflux of OTA. These data support the hypothesis that OAT4 and Oat5 are functional orthologs and share selectivity for OTA both for reabsorption and secretion. SIGNIFICANCE STATEMENT: This study compares the selectivity profile between human organic anion transporter (OAT4) and mouse Oat5. Our data revealed a similar selectivity profile for ochratoxin A (OTA) reabsorption and secretion by these two transporters, thereby supporting the hypothesis that hOAT4 and mOat5, although not genetic orthologs, behave as functional orthologs for both uptake and efflux. These data will be instrumental in selecting an appropriate animal model when studying the renal disposition of anionic drugs and toxicants.

Subchronic exposure of individual and combined ochratoxin A and citrinin selectively affects the expression of rat renal organic cation transporters.

Ochratoxin A (OTA) and citrinin (CIT) are nephrotoxins found co-occurring in various human/animal food/feed and recognized as a health threat. However, most studies investigate individual effects and neglect their combined nephrotoxic effects in mammals. Previous studies have indicated that organic anion/cation transporters (OATs/OCTs) localized in renal proximal tubules mediate the transport of OTA and CIT. Still, little is known about the in vivo effects of individual/combined OTA and CIT on protein localization/expression of OCTs, physiologically/pharmacologically important renal transporters. Here, we used Western blot and immunofluorescence microscopy to study the effects of subchronic (21-day) exposure to individual/combined OTA (0.125 and 0.250mgkg-1 b.w.) and CIT (20mgkg-1 b.w.) on protein localization/expression of organic cation transporters (rOct1/Slc22a1 and rOct2/Slc22a2) in kidneys of Wistar rats. Since the antioxidant resveratrol (RSV) has shown measurable protective effects against OTA- and CIT-related oxidative stress toxicity in vitro, we investigated the effects of an OTA + CIT + RSV combination on rOct1/2 localization/expression in the same model. Individual OTA induced a dose-dependent decrease of rOct1 but not rOct2 protein expression, whereas their localization pattern remained unchanged. Individual CIT did not affect the renal rOct1/2 protein localization/expression. Combined OTA + CIT exposure induced a significant decrease of rOct1 protein expression by an OTA250 dose, whereas oral co-administration of OTA + CIT + RSV resulted in a significant decrease of rOct1/2 protein expression. Thus, we revealed an OTA-related selective effect on the rOct1/2 protein expression and a non-specific adverse effect of RSV in the OTA + CIT + RSV combination on the renal organic cation transport system in rat.

Microphysiological system modeling of ochratoxin A-associated nephrotoxicity

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.

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

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 pH7.4 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 pH7.4, whereas both mBcrp and hBCRP clearly transported OTA at pH6.4. There was modest transport of OTA by mMrp2 and hMRP2 only at pH6.4. OATP1A2 and OATP2B1 mediated uptake of OTA both at pH7.4 and 6.4, but OATP1B1 only at pH7.4. 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.

The influence of displacement compounds on the binding of ochratoxin A to human serum albumin examined with fluorescence anisotropy methods

The subject of the research is the application of the methods of fluorescence anisotropy measurements to study the interaction between human serum albumin (HSA) and toxins and selected medicines (ibuprofen, warfarin, flurbiprofen). Optical spectroscopic methods are useful tools for the study of biologically active compounds. Determining binding intensity of ochratoxin A (OTA) to albumin may be helpful in explaining the effects of toxic influence of OTA. The main factor influencing the distribution of OTA is its affinity for plasma proteins. It was shown that ochratoxin binds strongly to albumin. By the use of the method of fluorescence anisotropy it was proved that the unbound fraction of OTA is higher due to competing interactions with drugs. As a result of separating ochratoxin from protein by competitive compounds, a decrease in the fluorescence anisotropy of the HSA-OTA complex was observed. The largest increase in free fraction of OTA is caused by flurbiprofen, then ibuprofen and warfarin. It will accelerate OTA transport to target organs and shortening its half-life period, leading consequently to a decrease in chronic toxic effects.

A review on ochratoxin A transcriptomic studies

The mycotoxin Ochratoxin A (OTA) is a potent renal carcinogen in male rats. Transcriptomic studies on OTA (4 in vitro, 6 in vivo, 2 in vitro/in vivo) have been reviewed. The aim of 6 of them was mainly mechanistic whereas the rest had mostly predictive (1) or evaluation (5) purposes. An overall tendency towards gene expression downregulation was observed, probably as a result of protein synthesis inhibition. DNA damage response genes were not deregulated in most of the studies. Genes involved in acute renal injury, cell survival and cell proliferation were upregulated in several in vivo studies. Apoptosis genes were deregulated in vitro but less affected in vivo; activation of several MAPKs has been observed. Many genes related to oxidative stress or involved in cell-to-cell interaction pathways (Wnt) or cytoskeleton structure appeared to be deregulated either in vitro or in vivo. Regucalcin was highly downregulated in vivo and other calcium homeostasis genes were significantly deregulated in vitro. Genes related to OTA transport (OATs) and metabolism (CYPs) appeared downregulated in vivo. Overall, the mechanism of action of OTA remains unclear, however transcriptomic data have contributed to new mechanistic hypothesis generation and to in vitro-in vivo comparison.

Fate of the teratogenic and carcinogenic ochratoxin A in human perfused placenta

Ochratoxin A (OTA) is one of the most frequent mycotoxins detected in human blood worldwide. Apart from its well known nephrotoxicity, OTA-induced teratogenicity and carcinogenicity proven in animals are potential effects also in humans. Pregnant women have been exposed to this food contaminant via dietary exposure in a continuous and widespread manner. Although the transplacental transfer of OTA has been demonstrated in laboratory animals and the presence of OTA in human fetal samples has been reported, little is known about the role of human placenta in OTA toxicokinetics. In this study, human perfused placenta was used to reveal the actual placental toxicokinetics of OTA using concentrations found in serum of pregnant women. Moreover, the effect of protein concentration and biological significance of placental transporters on the OTA transfer in human placenta were also determined. Our study is the first to pursue the transfer of OTA through perfused human placenta. The transfer of OTA through term human placenta was barely detectable in all perfusions. Inhibitors of neither ABCG2 nor ABCC2 increased the transport of OTA to fetal circulation in placental perfusion, and thus these transporters apparently do not have biological significance in inhibiting transplacental transfer of OTA. Human albumin has inhibited OTA transfer through a tight monolayer of BeWo b30 cells. Finding from this study clearly contradict the existing epidemiological studies reporting higher OTA levels in fetal than in maternal circulation in vivo.

Molecular Mechanism of Ochratoxin A Transport in the Kidney

The mycotoxin, ochratoxin A (OTA), is thought to be responsible for Balkan endemic nephropathy. OTA accumulates in several tissues, especially in the kidneys and liver. The excretion of OTA into urine is thought to be mainly by tubular secretion, presumably via the organic anion transport system. Recently, several families of multispecific organic anion transporters have been identified: organic anion transporters (OATs), organic anion-transporting polypeptides (OATPs), oligopeptide transporters (PEPTs), and ATP-binding cassette (ABC) transporters, such as MRP2 and BCRP. These renal transporters mediate the transmembrane transport of OTA and play a pivotal role in the development of OTA-induced nephrotoxicity.

New examinations of mycotoxin carryover to cocoa beans

Mycotoxins are secondary metabolites which can form on various foodstuffs through the growth of certain fungi. Ochratoxin A (OTA) and the aflatoxins B1 B2, G1 and G2 have been detected in low concentrations in cocoa and cocoa products. As regards the question of in what stages of the cocoa production process a contamination with the mycotoxin-producing moulds and the formation of mycotoxins takes place, it is assumed that in the case of cocoa the contamination is not concerning the individual beans but the fermentation units.A model test was carried out to provide information on the process by which a possible carryover of the above-mentioned mycotoxins to cocoa beans occurs during the fermentation process. For this purpose fresh cocoa beans were left to soak in an artificial mycotoxin-containing fermentation solution. The mycotoxin levels in the cocoa beans were regularly determined over a period of 12 days. New findings were made as regards the migration of mycotoxins during the fermentation process. We interpret the divergent uptake behaviour of the mycotoxins to indicate that the transport of OTA and that of aflatoxins does not take place in the same manner. This is possibly caused by chemico-physical effects, such as the different polarities of the mycotoxins.

Differential modulation of ochratoxin A absorption across Caco-2 cells by dietary polyphenols, used at realistic intestinal concentrations

The effect of polyphenols (PPs) on the absorption of ochratoxin A (OTA), a food-borne mycotoxin, was investigated in an in vitro model of the human intestinal barrier based on Caco-2 cells cultivated in a bicameral system. Two intraluminal concentrations of OTA approaching physiological levels were chosen (0.75 nM and 7.5 nM) through calculations based on estimated daily intakes. The transport of OTA from the apical to the basolateral side of Caco-2 cells, i.e. absorption, was directly proportional to its initial apical concentration. Very significant increase in both OTA absorption and cellular accumulation was observed upon co-incubation with certain PPs, i.e. chrysin, quercetin, genistein, biochanin A, resveratrol, at concentrations that should be encountered in the gastrointestinal tract, as well as with MK571, a specific inhibitor of MRPs efflux pumps. As these PPs have been reported to be metabolized in Caco-2 cells into substrates of MRP-2, we hypothesize that PPs and/or metabolites could impair the OTA efflux, previously proposed to be mediated by the MRP-2, through competitive inhibition for the pump. These data imply that interactions between OTA and PPs may lead to a greater bioavailability of the mycotoxin in the bloodstream with possible adverse effects for human health.

Molecular Aspects of the Transport and Toxicity of Ochratoxin A

AbstractFor Abstract see ChemInform Abstract in Full Text.

Molecular Aspects of the Transport and Toxicity of Ochratoxin A

Ochratoxins are a class of naturally occurring compounds produced by several fungi. The most toxic is ochratoxin A (OTA), and occurrence of some human nephropathies and tumors correlate with enhanced OTA exposure. In this Account, the following areas are examined: molecular details of the binding of OTA to human serum albumin (HSA), the influences of binding to HSA on the trans-port of OTA across epithelial cell membranes by organic anion transport proteins, the oxidative activation of OTA, and the formation of OTA adducts with biological molecules. These studies are beginning to provide a detailed chemical model for the trans-port, accumulation, and genotoxic and carcinogenic effects of OTA.

Interaction of ochratoxin A with human intestinal Caco-2 cells: possible implication of a multidrug resistance-associated protein (MRP2)

Ochratoxin A (OTA), a nephrotoxic mycotoxin, is absorbed from small intestine and, in plasma, binds to serum albumin. Prolonged half-live results from reabsorption by proximal tubules and enterohepatic circulation. The mechanism whereby OTA crosses intestine was investigated by means of a cell culture system consisting of Caco-2 cells, as in vitro model of human intestinal epithelium. Cytotoxicity assays on proliferating Caco-2 cells showed that 0.4 μM OTA inhibits MTT reduction by 50%. Transepithelial transport and intracellular accumulation of OTA were studied in Caco-2 cells, differentiated in bicameral inserts. At pH 7.4, OTA is transported preferentially in basolateral (BL) to apical (AP) direction, suggesting a net secretion. Conditions closer to in vivo situation in duodenum (AP pH 6.0, BL pH 7.4) increase intracellular accumulation and transepithelial transport. AP to BL transport becomes higher than BL to AP transport, suggesting OTA absorption. Addition of serum albumin in BL compartment further increases OTA absorption across Caco-2 cells and suggests that in vivo OTA transport from serosal to luminal side of enterocytes is prevented, due to its binding to plasma proteins. Competition experiments showed that carrier systems for large neutral amino acids, H +/dipeptides cotransporter, organic anion ( p-aminohippurate) carrier and organic anion transporter (oatp) are not implicated in OTA transport across Caco-2 cells, in contrast to what was reported in kidney and liver. AP and BL transport and intracellular accumulation of OTA are increased in the presence of non specific inhibitors of MRPs (indomethacin, genistein and probenecid) and of 1-chloro-2,4-dinitrobenzene (biotransformed into 2,4-dinitrophenyl-gluthatione, a specific inhibitor of MRPs), but are affected by verapamil, an inhibitor of P-gp. This suggests that the multidrug resistance-associated protein (MRP2) could be implicated in transepithelial transport. Therefore, absorption of OTA across the intestinal mucosa would be limited thanks to its excretion through MRP2 at the apical pole of enterocytes.

Role of human organic anion transporter 4 in the transport of ochratoxin A

The purpose of this study was to investigate the characteristics of ochratoxin A (OTA) transport by multispecific human organic anion transporter 4 (hOAT4) using mouse proximal tubule cells stably transfected with hOAT4 (S 2 hOAT4). Immunohistochemical analysis revealed that hOAT4 protein was localized to the apical side of the proximal tubule. S 2 hOAT4 expressed hOAT4 protein in the apical side as well as basolateral side and the cells were cultured on the plastic dish for experiments. S 2 hOAT4 exhibited a time- and concentration-dependent, and a saturable increase in OTA uptake, with an apparent K m value of 22.9±2.44 μM. The OTA uptakes were inhibited by several substrates for the OATs. Probenecid, piroxicam, octanoate and citrinin inhibited OTA uptake by hOAT4 in a competitive manner ( K i=44.4–336.4 μM), with the following order of potency: probenecid>piroxicam>octanoate>citrinin. The efflux of OTA by S 2 hOAT4 was higher than that by mock. Addition of OTA resulted in slight decrease in viability of S 2 hOAT4 compared with mock. These results indicate that hOAT4 mediates the high-affinity transport of OTA on the apical side of the proximal tubule, whereas the transport characteristics of OTA are distinct from those by basolateral OATs.

Characterization of ochratoxin A transport by human organic anion transporters

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 (S 2) cells from mice stably expressing hOAT1 and hOAT3 (S 2 hOAT1 and S 2 hOAT3). S 2 hOAT1 and S 2 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.

Apical-to-basolateral transepithelial transport of Ochratoxin A by two subtypes of Madin-Darby canine kidney cells

In this study we investigated the transepithelial transport of Ochratoxin A (OTA), a potent nephrotoxin, across monolayers of two collecting duct-derived cells clones (Madin-Darby canine kidney cells (MDCK)-C7 and MDCK-C11 cells, resembling principal and intercalated cells, respectively) either from the apical to the basolateral side or vice versa. We cultured cells on permeable supports and compared the transport rates of OTA, p-aminohippuric acid (PAH) and fluorescein-labelled inulin. Monolayers of both cell clones translocated OTA from the apical to the basolateral side but not in the opposite direction. Transport rate across MDCK-C11 cell monolayers was 2.9-fold the transport rate across MDCK-C7 cell monolayers. OTA transport was temperature-dependent being reduced from 77.5 pmol/cm 2 per h to 10.1 pmol/cm 2 per h in MDCK-C11 and from 27.0 pmol/cm 2 per h to 7.6 pmol/cm 2 per h in MDCK-C7 cells when temperature was decreased from 37°C to 4°C. In both cell clones, the dipeptides carnosine and glycylsarcosine but not the amino acids glycine or phenylalanine had an inhibitory effect on OTA transport. In both cell clones, transepithelial transport of OTA was dependent on the apical pH (p K a of OTA=7.1). In an environment mimicking the transepithelial in vivo pH gradient to some extent with more acidic pH on the apical side than on the basolateral side, transport was 4-fold higher in both cell clones as compared to conditions when pH was 7.4 in both bath solutions. In the absence of a pH gradient, transport rates were similar to that at 4°C. Apical uptake of [ 3H]OTA was inhibited by carnosine and by glycylsarcosine and the uptake of [ 3H]carnosine was inhibited by OTA. Our results indicate that OTA is transported across the apical membrane of MDCK cells by both non-ionic diffusion and by a H +-dipeptide cotransporter. Thus, reabsorption of OTA in the collecting duct contributes to the observed long half life of OTA in the mammalian body.