Inhibition of cytochrome P450 enhances the nephro- and hepatotoxicity of ochratoxin A

Abstract

The mycotoxin ochratoxin A (OTA) is a contaminant in food that causes nephrotoxicity and to a minor degree hepatotoxicity. Recently, we observed that OTA induces liver damage preferentially to the cytochrome P450 (CYP)-expressing pericentral lobular zone, similar to hepatotoxic substances known to be metabolically toxified by CYP, such as acetaminophen or carbon tetrachloride. To investigate whether CYP influences OTA toxicity, we used a single dose of OTA (7.5 mg/kg; intravenous) with and without pre-treatment with the pan CYP-inhibitor 1-aminobenzotriazole (ABT) 2 h before OTA administration. Blood, urine, as well as liver and kidney tissue samples were collected 24 h after OTA administration for biochemical and histopathological analyses. Inhibition of CYPs by ABT strongly increased the nephro- and hepatotoxicity of OTA. The urinary kidney damage biomarkers kidney injury molecule-1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL) were increased > 126-fold and > 20-fold, respectively, in mice treated with ABT and OTA compared to those receiving OTA alone. The blood biomarkers of liver damage, alanine transaminase (ALT) and aspartate transaminase (AST) both increased > 21- and 30-fold, respectively, when OTA was administered to ABT pre-treated mice compared to the effect of OTA alone. Histological analysis of the liver revealed a pericentral lobular damage induced by OTA despite CYP-inhibition by ABT. Administration of ABT alone caused no hepato- or nephrotoxicity. Overall, the results presented are compatible with a scenario where CYPs mediate the detoxification of OTA, yet the mechanisms responsible for the pericental liver damage pattern still remain to be elucidated.

Keywords: Bioactivation; Detoxification; Drug metabolism; Metabolic zonation; Mycotoxins.

Fig. 1

Biotransformation of ochratoxin A (OTA; central box) to metabolites identified in vivo and/or in vitro and proposed intermediates. Some pathways (green arrows) result in the formation of a product (OT-alpha) with relatively low toxicity and to conjugated metabolites which are more readily excreted than the parent mycotoxin. Formation of hydroxylated OTA metabolites by CYP450 enzymes (blue arrows) is also regarded as detoxication reaction. In contrast, CYP-mediated oxidation of OTA to quinone/hydroquinone intermediates (pink arrows) is considered as potential bioactivation reaction. The quinone/hydroquinone couple may react with cellular macromolecules or undergo conjugation by GSTs (grey arrows) to glutathione conjugates, e.g., the OTB-GSH metabolite which is then transformed into a mercapturic acid. Opening of the lactone ring of OTA (pink arrow to the top) yields a product with increased toxicity. See text for further details and references on OTA metabolism. A pink arrow pointing to a question mark denotes hypothetical as yet unknown bioactivation reactions for OTA

Fig. 2

CYP-inhibition by aminobenzotriazole (ABT) blocks acetaminophen (APAP) induced hepatotoxicity. A Macroscopical appearance of livers 6 h after an overdose of 300 mg/kg APAP alone or in combination with the non-specific CYP-inhibitor ABT (150 mg/kg; gavage). ABT was administered 2 h before APAP. The control groups received only vehicle (PBS) or ABT. B Plasma activities of the liver enzyme ALT and AST 6 h after APAP administration with and without ABT compared to vehicle or ABT controls; *p value ≤ 0.05, **p value ≤ 0.01 Tukey’s multiple comparisons test; n = 3 mice per group. C Histological appearance (H&E staining) of control (vehicle or ABT treated) and APAP-treated livers with and without ABT in 20-fold magnification with accompanying overview of entire liver lobules

Fig. 3

CYP-inhibition enhances the hepatotoxicity of ochratoxin A (OTA). A Macroscopic appearance of livers 24 h after administration of OTA (7.5 mg/kg; i.v.) with and without ABT (150 mg/kg, gavage; administered 2 h before OTA) compared to vehicle (PBS) or ABT controls. B Plasma activities of liver enzymes 24 h after OTA administration with and without ABT compared to vehicle or ABT controls; *p value ≤ 0.05, **p value ≤ 0.01 Tukey’s multiple comparisons test; n = 5 mice per group. C Histological appearance in H&E-stained tissue sections; scale bars: 50 µm

Fig. 4

Infiltration of immune cells into regions of pericentral tissue damage induced by OTA intoxication with and without ABT treatment compared to vehicle or ABT controls. A Immunostaining for the common leukocyte antigen CD45 and the macrophage marker FSP1 (B). The same experimental design as described in Fig. 3 was applied; scale bars: 10 µm (closeup) and 200 µm (overview)

Fig. 5

CYP-inhibition enhances the nephrotoxicity of OTA. A H&E staining; scale bars 50 µm. B CD45 immunostaining; scale bars 10 µm. C Serum creatinine and blood urea nitrogen. D NGAL and KIM-1 urinary concentrations 24 h after OTA administration with and without ABT compared to vehicle or ABT controls; **p value ≤ 0.01 Tukey’s multiple comparisons test; n = 5 mice per group. The same experimental design as in Fig. 3 was applied

Fig. 6

Treatment with ABT does not influence albumin expression in the liver as evidenced by immunostaining (A) and serum albumin (B); scale bars 50 µm; n = 5 mice per group. The mice received a dose of 150 mg/kg ABT by gavage and the analyses were performed 24 h later

Fig. 7

Treatment with ABT has no major influence on the transport of OTA from the sinusoidal blood into hepatocytes and secretion into the bile canaliculi. A Intravital imaging of OTA (7.5 mg/kg intravenous) transport in livers of mice pre-treated (2 h) with ABT (150 mg/kg, gavage) or vehicle. The time in the upper left gives the minutes after injection of OTA. OTA is visualized by its blue auto-fluorescence; the morphology of hepatocytes is visualized by the red dye TMRE. B and C Quantification of OTA-associated fluorescence in blood sinusoids, hepatocytes and in bile canaliculi in controls (vehicle) and in ABT pre-treated mice. The corresponding time-lapse videos are available in the supplement. Three mice were imaged per group