Ozone aging amplifies the tumor-promoting effects associated with low-dose graphene oxide exposure in bladder tumors via the integrin αV signaling pathway.

With the rapid expansion of industrial and commercial applications, graphene-based 2D nanomaterials such as graphene oxide (GO) are increasingly released into the environment, raising concerns about their environmental accumulation and the likelihood of human exposure. While previous studies have primarily focused on the cytotoxic effects of pristine GO (P-GO) at high exposure doses, the biological effects of GO under real environmental conditions remain poorly understood. Notably, atmospheric environmental media, such as ozone, can significantly alter the physicochemical properties of GO, thereby modulating its biological effects. In this study, we employed a non-direct toxicity dose to demonstrate that ozone aging can significantly modify the physicochemical properties of GO and enhance its low-dose exposure-mediated pro-tumor progression effect. Mechanistically, ozone-aged GO (O-GO) treatment increases the membrane localization of integrin αV, leading to the activation of the PI3K/AKT/mTOR signaling pathway, which drives bladder tumor proliferation. Furthermore, O-GO enhances the TGF-β signaling pathway by upregulating the expression levels of the TGF-β receptor, promoting bladder tumor cell invasiveness and metastasis. These findings provide novel insights into the tumor-promoting effects of GO at non-direct toxicity dose and emphasize the necessity of considering environmental aging process when assessing its environmental health and safety (EHS) risks. Given the expanding use of graphene-based materials in daily human life, our study highlights the urgent need for greener development strategies to mitigate potential health risks associated with GO exposure.