The neuronal nitric oxide synthase inhibitor NANT blocks acetaminophen toxicity and protein nitration in freshly isolated hepatocytes

Abstract

3-Nitrotyrosine (3NT) in liver proteins of mice treated with hepatotoxic doses of acetaminophen (APAP) has been postulated to be causative in toxicity. Nitration is by a reactive nitrogen species formed from nitric oxide (NO). The source of the NO is unclear. iNOS knockout mice were previously found to be equally susceptible to APAP toxicity as wildtype mice and iNOS inhibitors did not decrease toxicity in mice or in hepatocytes. In this work we examined the potential role of nNOS in APAP toxicity in hepatocytes using the specific nNOS inhibitor NANT (10 µM)(N-[(4S)-4-amino-5-[(2-aminoethyl)amino]pentyl]-N'-nitroguanidinetris (trifluoroacetate)). Primary hepatocytes (1 million/ml) from male B6C3F1 mice were incubated with APAP (1mM). Cells were removed and assayed spectrofluorometrically for reactive nitrogen and oxygen species using diaminofluorescein (DAF) and Mitosox red, respectively. Cytotoxicity was determined by LDH release into media. Glutathione (GSH, GSSG), 3NT, GSNO, acetaminophen-cysteine adducts, NAD, and NADH were measured by HPLC. APAP significantly increased cytotoxicity at 1.5-3.0 h. The increase was blocked by NANT. NANT did not alter APAP mediated GSH depletion or acetaminophen-cysteine adducts in proteins which indicated that NANT did not inhibit metabolism. APAP significantly increased spectroflurometric evidence of reactive nitrogen and oxygen formation at 0.5 and 1.0 h, respectively, and increased 3NT and GSNO at 1.5-3.0 h. These increases were blocked by NANT. APAP dramatically increased NADH from 0.5-3.0 h and this increase was blocked by NANT. Also, APAP decreased the Oxygen Consumption Rate (OCR), decreased ATP production, and caused a loss of mitochondrial membrane potential, which were all blocked by NANT.

Keywords: 3-nitrotyrosine; Acetaminophen; Hepatotoxicity; Mitochondria; Nitric Oxide; Reactive nitrogen and oxygen species; S-nitrosoglutathione.

Figure 1

Effect of NANT on APAP-induced toxicity, glutathione, and covalent binding in freshly isolated hepatocytes. Hepatocytes were incubated with APAP (1 mM), APAP plus NANT (10 uM), NANT alone or with media alone (Con) for 0–3h. Time points were taken, (A) relative toxicity was measured using LDH release, (B) GSH, (C) APAP-Cys, and (D) GSSG in the hepatocytes. Significant increase was indicated by * (p≤0.05) from control group. Samples were n=3 from 3 separate mice and hepatocytes isolated on 3 different days. The data are presented as mean ± SE.

Figure 2

Effect of NANT on APAP-induced reactive oxygen and nitrogen formation in freshly isolated hepatocytes. Hepatocytes were incubated with APAP (1mM), APAP plus NANT (10uM), NANT alone or with media alone (Con) for 0–3 h. Time points were taken, (A) 3-NT in proteins, (B) Nitric Oxide production (C) Reactive Oxygen production, and (D) GSNO in the hepatocytes. Significant increase was indicated by * (p≤0.05) from control group. Samples were n=3 from 3 separate mice and hepatocytes isolated on 3 different days. The data are presented as mean ± SE

Figure 3

Effect of NANT on APAP-induced NAD and NADH levels in freshly isolated hepatocytes. Hepatocytes were incubated with APAP (1 mM), APAP plus NANT (10 uM), NANT alone and with media alone (Con) for 0–3h. Time points were taken, (A) NADH and (B) NAD⁺ in the hepatocytes. Significant increase was indicated by * (p≤0.05) from control group. Samples were n=3 from 3 separate mice and hepatocytes isolated on 3 different days. The data are presented as mean ± SE.

Figure 4

Effect of NANT on APAP-induced alterations of oxygen consumption, mitochondrial membrane potential, and ATP levels in freshly isolated hepatocytes. Hepatocytes were incubated with APAP (1 mM), APAP plus NANT (10 uM), NANT alone and with media alone (Con) for 0–3 h. OCR (A) in the above mention groups were measured using Seahorse XF96 analyzer 1 in the graph represent the time at which APAP is injected in the groups and NANT in the groups. Time points were taken, (B) relative mitochondrial membrane potential and (C) ATP production in the hepatocytes. Significant increase was indicated by * (p≤0.05) from control group. Samples were n=3 from 3 separate mice and hepatocytes isolated on 3 different days. The data are presented as mean ± SE.

Figure 5

Postulated mechanism of APAP mediated mitochondrial toxicity by RNS.