Caffeine disrupts ataxia telangiectasia mutated gene-related pathways and exacerbates acetaminophen toxicity in human fetal immortalized hepatocytes

Preterm infants are at greater risk for adverse drug effects due to hepatic immaturity. Multiple interventions during intensive care increases potential for drug interactions. In this setting, high-dose caffeine used for apnea in premature infants may increase acetaminophen toxicity by inhibiting ataxia telangiectasia mutated (ATM) gene activity during DNA damage response. To define caffeine and acetaminophen interaction, we modeled infantile prematurity in late-gestation fetal stage through human immortalized hepatocytes and liver organoids. The acute toxicity studies included assays for cell viability, mitochondrial dysfunction and ATM pathway-related DNA damage. Fetal cells expressed hepatobiliary properties, albeit with lower metabolic, synthetic and antioxidant functions than more mature hepatocytes. Acetaminophen in IC50 amount of 7.5 millimolar caused significant oxidative stress, mitochondrial membrane potential impairments, and DNA breaks requiring ATM-dependent repair. Caffeine markedly exacerbated acetaminophen toxicity by suppressing ATM activity in otherwise nontoxic 2.5 millimolar amount. Similarly, the specific ATM kinase antagonist, KU-60019, reproduced this deleterious interaction in 5 micromolar amount. Replicative stress from combined acetaminophen and caffeine toxicity depleted cells undergoing DNA synthesis in S phase and activated checkpoints for G0/G1 or G2/M restrictions. Synergistic caffeine and acetaminophen toxicity in liver organoids indicated these consequences should apply in vivo. The antioxidant, N-acetylcysteine, decreased oxidative damage, mitochondrial dysfunction and ATM pathway disruption to mitigate caffeine and acetaminophen toxicity. We concluded that hepatic DNA damage, mitochondrial impairment and growth-arrest after combined caffeine and acetaminophen toxicity will be harmful for premature infants. Whether caffeine and acetaminophen toxicity may alter outcomes in subsequently encountered hepatic disease needs consideration.