Exposure time to caffeine affects heartbeat and cell damage-related gene expression of zebrafish Danio rerio embryos at early developmental stages

Caffeine is white crystalline xanthine alkaloid that is naturally found in some plants and can be produced synthetically. It has various biological effects, especially during pregnancy and lactation. We studied the effect of caffeine on heartbeat, survival and the expression of cell damage related genes, including oxidative stress (HSP70), mitochondrial metabolism (Cyclin G1) and apoptosis (Bax and Bcl2), at early developmental stages of zebrafish embryos. We used 100μm concentration based on the absence of locomotor effects. Neither significant mortality nor morphological changes were detected. We monitored hatching at 48h post-fertilization (hpf) to 96hpf. At 60 and 72hpf, hatching decreased significantly (P<0.05); however, the overall hatching rate at 96hpf was 94% in control and 93% in caffeine treatment with no significant difference (P>0.05). Heartbeats per minute were 110, 110 and 112 in control at 48, 72 and 96hpf, respectively. Caffeine significantly increased heartbeat - 122 and 136 at 72 and 96hpf, respectively. Quantitative RT-PCR showed significant up-regulation after caffeine exposure in HSP70 at 72hpf; in Cyclin G1 at 24, 48 and 72hpf; and in Bax at 48 and 72hpf. Significant down-regulation was found in Bcl2 at 48 and 72hpf. The Bax/Bcl2 ratio increased significantly at 48 and 72hpf. We conclude that increasing exposure time to caffeine stimulates oxidative stress and may trigger apoptosis via a mitochondrial-dependent pathway. Also caffeine increases heartbeat from early phases of development without affecting the morphology and survival but delays hatching. Use of caffeine during pregnancy and lactation may harm the fetus by affecting the expression of cell-damage related genes.