Increased susceptibility to methotrexate-induced toxicity in nonalcoholic steatohepatitis

Abstract

Hepatic drug metabolizing enzymes and transporters play a crucial role in determining the fate of drugs, and alterations in liver function can place individuals at greater risk for adverse drug reactions (ADRs). We have shown that nonalcoholic steatohepatitis (NASH) leads to changes in the expression and localization of enzymes and transporters responsible for the disposition of numerous drugs. The purpose of this study was to determine the effect of NASH on methotrexate (MTX) disposition and the resulting toxicity profile. Sprague Dawley rats were fed either a control or methionine-choline-deficient diet for 8 weeks to induce NASH, then administered a single ip vehicle, 10, 40, or 100 mg/kg MTX injection followed by blood, urine, and feces collection over 96 h with terminal tissue collection. At the onset of dosing, Abcc1-4, Abcb1, and Abcg2 were elevated in NASH livers, whereas Abcc2 and Abcb1 were not properly localized to the membrane, similar to that previously observed in human NASH. NASH rodents receiving 40-100 mg/kg MTX exhibited hepatocellular damage followed by initiation of repair, whereas damage was absent in controls. NASH rodents receiving 100 mg/kg MTX exhibited slightly greater renal toxicity, indicating multiple organ toxicity, despite the majority of the dose being excreted by 6 h. Intestinal toxicity in NASH however, was strikingly less severe than controls, and coincided with reduced fecal MTX excretion. Because MTX-induced gastrointestinal toxicity limits the dose escalation necessary for cancer remission, these data suggest a greater risk for life-threatening MTX-induced hepatic and renal toxicity in NASH in the absence of overt gastrointestinal toxicity.

Keywords: ABC transporters; adverse drug reactions; hepatobiliary disposition; hepatotoxicity; methotrexate.

FIG. 1.

Hepatic transporter expression and localization. (A) Representative immunoblots of Abcc1–4, Abcb1, Abcg2, and total ERK in control and MCD rodents with two samples from each treatment group. (B) Relative protein levels of all samples normalized to total ERK; mean ± SEM. IHC staining of Abcc2 (C) and Abcb1 (D), 100× objective magnification. Asterisks (*) indicate p ≤ 0.05, control versus MCD; daggers (†) indicate p ≤ 0.05, MTX dose versus vehicle. Arrows indicate disrupted transporter localization.

FIG. 2.

MTX-induced hepatocellular damage. Representative images of (A) H&E-stained liver, (B) IHC staining of PCNA, and (C) Masson Trichrome staining of fibrosis; objective magnification is as indicated. Arrows indicate biliary hyperplasia (A), positive PCNA staining (B), and fibrosis (C).

FIG. 3.

Renal transporter expression and MTX-induced kidney damage. (A) Representative immunoblots of Abcc2, Abcc3, Abcg2, and total ERK in control and MCD rodents with two samples from each treatment group. (B) Relative protein levels of all samples normalized to total ERK; mean ± SEM. Representative H&E-stained kidney (C), 20× objective magnification. Asterisks (*) indicate p ≤ 0.05, control versus MCD; daggers (†) indicate p ≤ 0.05, MTX dose versus vehicle. Arrows indicate protein cast formation.

FIG. 4.

High-dose MTX-induced intestinal damage. Representative images of H&E-stained (A) duodenum, jejunum, ileum, and (B) proximal and distal colon in control and MCD rodents, 20× objective magnification.

FIG. 5.

MTX disposition. MTX levels in (A) plasma, (B) feces, and (C) urine at indicated time points; mean ± SEM. (D) Cumulative total urinary MTX. Asterisks (*) indicate p ≤ 0.05, control versus MCD.