Podophyllotoxin induces testicular toxicity via the microbiota-gut-testis axis based on the toxicological evidence chain concept.

Podophyllotoxin (PPT), a natural lignan with well-established antitumor efficacy and well-documented ovarian toxicity, was first investigated for its potential testicular toxicity in male Sprague-Dawley rats, following the guidance of the Toxicological Evidence Chain (TEC) framework. Through an integrated approach combining physiological and pathological phenotyping, targeted metabolomics of intestinal contents, serum, and testicular tissues, testicular transcriptomics, and 16S rDNA sequencing of gut microbiota, we systematically delineated the injury evidence chain induced by PPT. Oral administration of PPT (20 mg/kg/day for 4 days) elicited marked systemic toxicity, including reduced body weight and food intake, alongside clinical signs such as nasal hemorrhage and diarrhea. Testicular toxicity was evident from decreased testicular weight and organ index, histopathological impairments-including seminiferous tubule distortion, fibrosis, and germ cell apoptosis-and compromised sperm quality. Serum levels of testosterone (T), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) were significantly suppressed. Concurrently, colonic injury was observed alongside gut dysbiosis, characterized by a reduction in Lactobacillus and enrichment of Proteus and Escherichia-Shigella. Multi-tissue metabolomics consistently identified disruption of vitamin B6 metabolism, with downregulation of pyridoxine, pyridoxamine, and 4-pyridoxic acid, while transcriptomic analysis revealed suppression of steroidogenic genes (Star, Cyp11a1, Cyp17a1). These findings collectively indicate that PPT induces testicular injury via the microbiota-gut-testis (MGT) axis.