S-Methylmethionine Attenuates Aflatoxin B1-Induced Mammary Epithelial Injury: Functional Assessment and Exploratory ATAC-Seq/RNA-Seq Analysis

Aflatoxin 1. (AFB1) threatens animal health and dairy safety, but mammary-protective interventions remain poorly defined. We evaluated whether S-methylmethionine (SMM; commonly known as vitamin U) attenuates AFB1-associated injury using MAC-T bovine mammary epithelial cells, a mouse mammary model, biochemical assays, RT-qPCR, Western blotting, H&E staining, and exploratory ATAC-seq/RNA-seq analysis. Under 4 μg/mL (approximately 12.81 μM) AFB1 challenge, 1 mM SMM increased MAC-T cell viability by 14.1% (Tukey-adjusted p = 0.0387) and reduced LDH release by 38.2% (p = 0.000236) relative to the AFB1-only group. SMM also shifted selected redox markers toward a less injured state. Nrf2 protein abundance differed between the AF and AU groups (p = 0.049), although pathway activation and causal dependence were not tested. In mice, SMM co-treatment reduced serum ALT by 38.5% and mammary MDA by 56.4%, while increasing mammary T-SOD by 85.4% and GSH-Px by 100.9% relative to AF (p < 0.05 for the reported pairwise comparisons); inflammatory transcripts changed non-uniformly. The AU-versus-AF integration yielded four exploratory candidate genes (TNFSF10, NBEAL1, ENSBTAG00000054644, and ENSBTAG00000052086), and the selected RT-qPCR results were directionally consistent with the RNA-seq data. Thus, SMM attenuated selected AFB1-associated injury features under the tested conditions, while the underlying mechanism remains unresolved.