Effect Of 1-aminobenzotriazole Research Articles

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.

Differential Modulation of Cytochrome P450 Activity and the Effect of 1-Aminobenzotriazole on Hepatic Transport in Sandwich-Cultured Human Hepatocytes

Sandwich-cultured human hepatocytes (SCHH) have been widely used for in vitro assessments of biliary clearance. However, the modulation of metabolism enzymes has not been fully evaluated in this system. The present study was therefore undertaken to determine the activity of cytochrome P450 (P450) 1A2, 2C8, 2C9, 2C19, 2D6, and 3A and to evaluate the impact of 1-aminobenzotriazole (ABT) on hepatic uptake and biliary excretion in SCHH. The SCHH maintained integrity and viability as determined by lactate dehydrogenase release and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assays conducted over the culture period. Although all assessed P450 activity decreased in day 2 SCHH, the extent of the decrease and the subsequent rebound in activity varied across the different isoforms. Day 5 CYP1A2 activity was approximately 2.5-fold higher than day 1 activity, whereas the CYP3A and CYP2C9 activities were 90 and 60% of the day 1 levels, respectively. In contrast, the initial CYP2C8, CYP2C19, and CYP2D6 activity losses did not rebound over the 5-day culture period. Furthermore, ABT was not found to have an effect, whether directly or indirectly as a P450 inactivator, with respect to the hepatic transport of rosuvastatin, atrovastatin, and midazolam in SCHH. Taken together, these results suggest that the SCHH model is a reliable tool to characterize hepatic uptake and biliary excretion. Due to the differential modulation of P450 activity, SCHH may not be considered a suitable tool for metabolic stability assessments with compounds predominantly cleared by certain P450 enzymes.

Effect of 1-aminobenzotriazole on the in vitro metabolism and single-dose pharmacokinetics of chlorzoxazone, a selective CYP2E1 substrate in Wistar rats

The aim of this study was to study the effect of 1-aminobenzotriazole (ABT) on in vitro metabolism, oral, and intravenous (IV) pharmacokinetics of chlorzoxazone (CZX) in rats. Enzyme kinetics of CZX was performed with rat and human liver microsomes and pure isozyme (CYP2E1) with and without ABT. The enzyme kinetics (Vmax and Km) of the formation of 6-hydroxychlorzoxazone (OH-CZX) was found to be similar among rat liver microsomes (3486 pmol mg protein−1 min−1 and 345 µM), human liver microsomes (3194 pmol mg protein−1 min−1 and 335 µM) and pure isozyme (3423 pmol mg protein−1 min−1 and 403 µM), but KI and Kinact values for ABT towards the ability to inhibit the formation of OH-CZX from CZX varied between liver microsomes (rat: 32.09 µM and 0.12 min−1; human: 27.19 µM and 0.14 min−1) and pure isozyme (3.18 µM and 0.29 min−1). The novel robust analytical method was capable of quantifying CZX, OH-CZX, and ABT simultaneously in a single run, and the method was used for both in vitro and in vivo studies. Pre-treatment of rats with ABT prior to oral and IV administration of CZX significantly decreased the clearance (threefold) and consequently increased the AUC of CZX (approx. three- to fourfold). When rats were pre-treated with ABT, the formation of OH-CZX was completely blocked after oral and IV administration; however, we were able to measure OH-CZX in rats administered with CZX by oral and IV routes without pre-treatment of ABT. The oral bioavailability of CZX was ∼71% when dosed alone and reached 100% under pre-treatment with ABT. The t1/2 values of CZX was significantly prolonged for oral dosing compared with IV dosing under pre-treated conditions with ABT, suggesting an involvement of pre-systemic component in the disposition of CZX. The pharmacokinetic parameters of ABT did not change when it was dosed along with CZX (oral and IV), indicating that either CZX or OH-CZX had no effect on disposition of ABT. The plasma concentrations of ABT were above and beyond the required levels to inhibit CYP2E1 enzyme for at least 36 h post-treatment.

Role of cytochrome P-450 in schisandrin B-induced antioxidant and heat shock responses in mouse liver

In order to explore the role of cytochrome P-450 ( P-450) in schisandrin B (Sch B)-induced antioxidant and heat shock responses, the effect of 1-aminobenzotriazole (ABT, a broad spectrum inhibitor of P-450) on hepatic mitochondrial glutathione antioxidant status (mtGAS) and heat shock protein (Hsp)25/70 expression was examined in Sch B-treated mice. The non-specific and partial inhibition of cytochrome P-450 ( P-450) by ABT pretreatment significantly caused a protraction in the time-course of Sch B-induced enhancement in hepatic mitGAS and Hsp25/70 expression in mice. Using mouse liver microsomes as a source of P-450, Sch B, but not dimethyl diphenyl bicarboxylate (a non-hepatoprotective analog of Sch B), was found to serve as a co-substrate for the P-450-catalyzed NADPH oxidation reaction, with a concomitant production of oxidant species. Taken together, the results suggest that oxidant species generated from P-450-catalyzed reaction with Sch B may trigger the antioxidant and heat shock responses in mouse liver.

Inhibition of Testicular Steroid Metabolism by Administration of 1-Aminobenzotriazole to Rats

Effects of 1-aminobenzotriazole (ABT) on testicular steroid metabolism were evaluated in rats. Administration of ABT to adult male rats caused dose-dependent decreases in testicular microsomal and mitochondrial cytochrome P450 concentrations. Significant losses of P450 occurred within 8 h of ABT treatment. Accompanying the declines in testicular P450 content were decreases in microsomal 17α-hydroxylase and mitochondrial cholesterol sidechain cleavage activities. Incubation of testicular microsomes or mitochondria in vitro with ABT plus an NADPH-generating system had no effect on P450 concentrations or on rates of steroid metabolism. By contrast, incubation of hepatic microsomes with ABT under the same conditions decreased P450 levels and xenobiotic-metabolizing activity. The results indicate that ABT in vivo causes inactivation of steroidogenic P450 isozymes in the testis, but the mechanism of inactivation differs from that on xenobiotic-metabolizing isozymes.

The influence of cytochrome P450 enzyme activity on the composition and quantity of volatile organics in expired breath

Abstract We have previously described a method to capture, identify and quantify volatile components in expired breath. The purpose of this research is to provide a non-invasive means to measure biomarkers of metabolism in vivo. In the present studies, the effect of 1-aminobenzotriazole (ABT), an inhibitor of diverse cytochrome P450 (P450) enzymes, on the composition of volatile organic chemicals (VOCs) expired in the breath of male F-344 rats was determined in parallel with the catalytic activities and total content of hepatic P450. lntraperitoneal administration of ABT (100 mg kg-(1)) to rats resulted in markedly diminished hepatic microsomal P450 content and activities. The extent of inhibition was near maximal at 4 h, at which time approximately 50% of the total P450 content, about 65% of the CYPlA2 activity, 55% of the CYP2E1 activity, and about 80% of CYP2B activity were lost. Inhibition was maintained to 48 h post-dosing, but P450 content and activities had largely been restored by day 7. Concomitant with the inhibition of P450 were corresponding increases (up to several hundred-fold) in the molar amount of volatiles appearing in the breath of ABT-treated animals, and the rebound of P450 levels was attended by corresponding decreases in the appearance of breath volatiles. These studies indicate that P450 plays a major role in the metabolism of VOCs appearing in breath, and that these chemicals can serve as markers on P450 activity in vivo.