Antioxidant pre-treatment prevents omeprazole-induced toxicity in an in vitro model of infectious gastritis

Abstract

Omeprazole is a mainstay of therapy for gastroesophageal reflux disease (GERD) and gastritis, and is increasingly used as an over-the-counter remedy for dyspepsia. Omeprazole acts by selectively oxidizing thiol targets in the gastric proton pump, but it also appears to be toxic to the gastric mucosa. We hypothesized that omeprazole toxicity is due to non-specific oxidation of cell structures other than the proton pump, and tested the efficacy of antioxidants to prevent omeprazole-induced toxicity in isolated rabbit gastric glands. Toxicity was measured by uptake and converstion of calcein-AM, following three hours of exposure to omeprazole and a non-selective thiol-oxidant, monochloramine. Intracellular concentration of Zn(2+) and the capacity to maintain luminal acidity were monitored using the fluorescent reporters fluozin-3 and Lysosensor DND-160, respectively. Both omeprazole and monochloramine caused marked reduction in cell viability. The toxicity of omeprazole was independent of monochloramine toxicity. The thiol reducing agent dithiothreitol protected gastric glands from injury. The oxidant scavenger Vitamin C also protected, and did not impair the anti-secretory effects of omeprazole. Thus, omeprazole toxicity appears to be oxidative and preventable with antioxidant therapy, including Vitamin C. Vitamin C may be a safe and efficacious addition to treatments requiring the use of PPIs.

Figure 1. Toxicity of omeprazole and monochloramine (NH₂Cl) as measured by calcein-AM uptake/conversion to fluorescent calcein

Glands were exposed to omeprazole (100µM) with or without NH₂Cl (0, 50, 100, 200µM) for 2.5 hours at 37°. Gland viability was assessed by uptake of calcein and its conversion of fluorescent calcein. Calcein-AM (8µM) was added for 30 minutes, and fluorescence was measured (Excitation 485, Emission 528). Data are expressed as % of Ringer’s control, mean ± SEM, N = 8. * = P < 0.05 vs. Ringer’s control. † P < 0.05 vs. 0 µM NH₂Cl.

Figure 2. Representative recording of [Zn²⁺] in an individual gastric gland, during exposure to omeprazole

Glands were loaded with Fluozin-3 AM (8µM, 30 minutes), placed on coverslips for real-time imaging, then equilibrated initially by perfusion with Ringer’s solution containing no added Ca²⁺ or Zn²⁺ and 0.5mM EGTA. Ringer’s solution containing omeprazole (100µM) was added after approximately 5 minutes. Each line represents a separate parietal cell in the gland, fluorescence in each cell being normalized to its baseline at the beginning of the experiment. Similar results were observed in three other glands.

Figure 3. Effect of pretreatment with BAPTA-AM on omeprazole and NH₂Cl toxicity

Glands were pre-treated with BAPTA-AM for 45 minutes, then exposed to omeprazole (100µM) and NH₂Cl for three hours. Gland viability was assessed by uptake of calcein and its conversion of fluorescent calcein. Data are expressed as % of Ringer’s control, mean ± SEM, N = 8. * = P < 0.05 vs. Ringer’s control. † P < 0.05 vs. 0 µM NH₂Cl.

Figure 4. Effect of TPEN on toxicities associated with omeprazole and NH₂Cl

Glands were co-incubated with omeprazole (100µM), NH₂Cl, and TPEN (20µM) for three hours. Gland viability was assessed by uptake of calcein and its conversion of fluorescent calcein. Data are expressed as % of Ringer’s control, mean ± SEM, N = 8. * = P < 0.05 vs. Ringer’s control. † P < 0.05 vs. 0 µM NH₂Cl.

Figure 5. Thiol reducing agent DTT (dithiothreitol) prevents both monochloramine and DTT damage

Glands were co-incubated with omeprazole (100µM), NH₂Cl, and DTT (1mM) for three hours. Gland viability was assessed by uptake of calcein and its conversion of fluorescent calcein. Data are expressed as % of Ringer’s control, mean ± SEM, N = 8. * = P < 0.05 vs. Ringer’s control. † P < 0.05 vs. 0 µM NH₂Cl.

Figure 6. Oxidant scavenger Vitamin C prevents omeprazole toxicity

Glands were co-incubated with omeprazole (100µM), NH₂Cl, and Vitamin C (1mM) for three hours. Gland viability was assessed by uptake of calcein and its conversion of fluorescent calcein. Data are expressed as % of Ringer’s control, mean ± SEM, N = 8. * = P < 0.05 vs. Ringer’s control. † P < 0.05 vs. 0 µM NH₂Cl.

Figure 7. Effects of Vitamin C effect on acidity in the tubulovesicle/lumen compartment of isolated gastric glands

Glands were co-incubated with omeprazole (100µM), NH₂Cl, and Vitamin C (1mM) for three hours. Lysosensor DND-160 (8µM) was added for 15 minutes, following which fluorescence was measured ratiometrically (Ex. 340nm/ 400nm, Em. 528nm). Data are expressed as the ratio of 340/400 excitations, mean ± SEM, N = 9. * = P < 0.05 vs. Ringer’s control. † P < 0.05 vs. Vitamin C (1mM) in Ringer’s.