Anticancer therapeutic efficacy of PEGylated β-galactosidase from Aspergillus terreus in DMBA-induced breast cancer: Toxicological, molecular and histopathological evaluation in Wistar rats.

Breast cancer remains a leading cause of cancer-related morbidity and mortality among women worldwide, underscoring the need for safer and more effective therapeutic strategies. Polyethylene glycol (PEG) conjugation is a well-established approach to enhance the stability, bioavailability, and in vivo performance of protein/enzymatic therapeutics. To our knowledge, this is the first study to demonstrate PEG conjugation of β-galactosidase and to provide in vivo evidence of its antitumor efficacy. Here, β-galactosidase from Aspergillus terreus was PEGylated using mPEG-NHS (5 kDa) and conjugation was validated by HPLC (retention time shift) and FTIR spectroscopy (PEG-associated ether bands with preserved protein amide signatures). Safety was evaluated following ARRIVE and OECD acute oral toxicity (TG 423) and 28-day repeated-dose oral toxicity (TG 407) protocols, showing no mortality, overt clinical toxicity, or treatment-related changes in hepatic or renal biochemical indices across the tested doses. Antitumor efficacy was assessed in a 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinoma model in female Wistar rats. Oral administration of PEGylated β-galactosidase (50 mg/kg for 30 days) attenuated DMBA-associated metabolic and oxidative disturbances, reflected by reduced lipid peroxidation (malondialdehyde) and restoration of enzymatic (SOD, CAT, GPx, GR, GST) and non-enzymatic (GSH, vitamins C and E) antioxidant defenses. Treatment further normalized membrane-bound ATPase activities, reduced tumor-associated enzyme markers (5'-nucleotidase, γ-glutamyltransferase, cathepsin D), regulated apoptosis-related endpoints (BAX, BCL-XL, P53; caspase-3) and improved mammary tissue histoarchitecture. Collectively, these findings support PEGylated β-galactosidase as a well-tolerated macromolecular candidate with in vivo antitumor potential mediated primarily through redox restoration and apoptosis regulation.