For the first 7 days, animals received regular drinking water. Caffeine (Sigma, St. Louis, MO, USA) was then administered in the drinking water at concentrations of 0.3, 0.6, 0.9, and 1.2 mg/ml, each dose for 7 days (the equivalent of ∼5–20 cups of coffee/day in humans when accounting for differences in liver size and metabolism (44)). After caffeine administration, the animals were switched back to regular water, and an additional 7 days (washout period) were recorded. In separate experiments (n = 4), mice received only a single dose of caffeine (0.6 mg/ml) for 7 days followed by washout for 7 days. To verify that water intake remained the same, water bottles were weighed throughout the experiment. We found no differences in average water intake over the entire experiment (Avg 24–33 ml/week; P = 0.59, Mann Whitney U test).”
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