The effects of early postnatal alcohol exposure on bone molecular composition in a mouse model

Background: Perinatal exposure to alcohol is a critical risk factor for long- term skeletal health in humans and has been demon strated in rodent models. Early postnatal alcohol gavage exposure in mice is used to study third trimester toxicity in humans. However, further research is required to fully characterize the effects of alcohol on bone molecular composition and distinguish them from the alterations associated with gavage- related stress in mouse models. Methods: Female mouse pups were assigned to untreated control (C), gavage control (GC), and alcohol- treated (A) groups. Between postnatal days 3 and 20, the A group received ethanol (3.0 g/kg/day) by intragastric gavage, while the GC group under went the same gavage procedure without any solution (neither milk nor ethanol). At postnatal day 90, femoral bone molecular composition and bone metabolism were evaluated by attenuated total reflection Fourier- transform infrared (ATR- FTIR) spec troscopy and biochemical studies. Results: Several ATR- FTIR- derived matrix and mineral- related alterations, including reduced total protein- related indices, lower collagen cross- link ratio, reduced mineral- to- matrix ratio, and increased crystallinity, were observed in both GC and A groups rel ative to untreated controls, indicating a substantial long- term influence of the gavage procedure itself. Compared with the GC, the A group showed additional selective differences, most notably reduced relative carbonate content and increased CTX- I levels. In the C group, serum 25(OH)D, PTH, and calcitonin levels differed from both GC and A groups, whereas osteocalcin was increased in the GC group relative to the C group, and no significant difference was observed between the GC and A groups. Conclusion: Prolonged neonatal gavage was associated with substantial long- term changes in bone molecular composition and several circulating bone- related hormonal markers. Against this background, postnatal alcohol exposure was associated with additional selective differences, particularly in CTX- I and relative carbonate content. These findings highlight the importance of discriminating between the effect of the procedure and the effect of alcohol in developmental models based on repeated neonatal gavage.