Prolonged high-dose prenatal caffeine exposure induces organ-specific developmental toxicity: renal p53 downregulation and hepatic caspase-3 upregulation in neonatal mice, supported with in silico studies

Abstract

Caffeine intake during pregnancy is widespread, yet it continues to raise public health concerns due to its implications for embryogenesis. Despite extensive research, the long-term consequences of prolonged high-dose caffeine exposure on critical phases of fetal organogenesis remain incompletely characterized. This study was aimed at investigating the impact of prolonged prenatal caffeine administration on kidney and liver development in Swiss albino mice. Pregnant mice (n = 18) received daily IP injections of caffeine (90 mg/kg/day) or saline from GD 8.5 to 18.5. Neonatal offspring were assessed for growth parameters, and molecular as well as histological analyses were performed on kidney and liver tissues, focusing on apoptosis-related markers p53 and caspase-3. Caffeine-exposed offspring showed significant growth restriction with reduced body size and weight and shorter crown-rump length. In the kidneys, caffeine caused marked suppression of p53 expression, a 3.5-fold increase in caspase-3 activity, and significant tubular disorganization. The liver maintained p53 expression but exhibited a significant increase in caspase-3 protein. Our in silico transcriptomic analysis revealed caffeine-induced dysregulation of cholesterol biosynthesis pathways in liver cells and ribosome biogenesis in renal cells, identifying key hub genes linked to lipid metabolism and RNA processing. These findings support our in vitro observations of p53 downregulation and caspase-3 activation, suggesting caffeine’s role in modulating apoptosis and cell cycle regulation. Prolonged high-dose caffeine exposure induces fetal growth impairment and organ-specific developmental toxicity, with different apoptotic dysregulation patterns in the kidneys by the p53-dependent pathway and in the liver by caspase-3-mediated pathways. This study highlights the importance of carefully considering caffeine dosage and duration of exposure during early pregnancy.