(290) Metabolomic Analysis to Elucidate the Mechanisms of Chemotherapy-induced Primary Ovarian Insufficiency

Metabolomic Analysis to Elucidate the Mechanisms of Chemotherapy-induced Primary Ovarian Insufficiency

Congcong Zhang¹, Wenlong Zhao¹, Shuo Xiao¹

1.  Department of Pharmacology and Toxicology, Ernest Mario School of Pharmacy, Environmental and Occupational Health Sciences Institute (EOHSI), Rutgers University, Piscataway, NJ 08854, USA

Abstract Text: Primary ovarian insufficiency (POI) and related early menopause and infertility are major side effects of cancer therapy in young female cancer patients. We previously used mouse models to demonstrate that doxorubicin (DOXO), a widely used chemo-drug, induces POI by activating DNA damage response (DDR) signaling in oocytes of primordial follicles, resulting in primordial follicle atresia and depletion of the ovarian reserve. However, the underlying mechanisms remain poorly understood. Prior research including our own has been focused on gene knockout mouse model, RNA sequencing (RNA-seq) analysis, and other molecular biology approaches. In this study, we used metabolomics, a powerful tool that unbiasedly measures cellular biochemical changes, to better understand the mechanisms of chemotherapy-induced POI. Postnatal day 5 (PND5) old CD-1 female mice were administrated with vehicle or a clinically relevant level of 10 mg/kg DOXO through intraparietal injection. At 6 hours post-injection, 30 ovaries from 15 mice were pooled as one replicate (n=8 replicates) to obtain ~10 mg proteins for metabolomic analysis using Liquid chromatography-high resolution mass spectrometry (LC-HRMS). Principal component analysis (PCA) separated vehicle and DOXO-treated ovaries into two clusters, indicating significantly changed metabotypes following DOXO treatment. Orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to extract candidate metabolites responsible for differentiating the DOXO-treated ovaries from the control. Model diagnostics using cross-validation and permutation confirmed the reliability and validity of OPLS-DA. The correlation coefficient of the absolute value＞0.514 and Variable Importance in Projection (VIP) > 1 were used to define differential metabolites between two groups. OPLS-DA using the negative ion mode identified 34 differential metabolites, with 24 up- and 10 down-regulated. In the positive ion mode, there were 26 differential metabolites, including 20 up- and 6 down-regulated, among which 14 up- and 1 down-regulated metabolites were not detected in the negative ion mode. Compared to the control, the top 5 up-regulated metabolites in DOXO-treated ovaries included Oxidized Nicotinamide Adenine Dinucleotide (NAD+), Deoxyinosine, AMP, Adenosine, and Glutamine. The top 5 down-regulated metabolites were Uridine, Xanthosine-5-phosphate, Thiamine, Biotin, Cytidine, and Hypoxanthine. Enrichment analysis revealed that the top 5 altered metabolic pathways were arginine synthesis, purine metabolism, pyrimidine metabolism, amino acid metabolism (glutamine metabolism, butanoate metabolism), and nicotinate-nicotinamide metabolism. NAD+ was the most up-regulated metabolite in DOXO-treated ovaries, which suggests the activation of poly (ADP-ribose) polymerase (PARP)-mediated DDR following DOXO treatment. Notably, prior research reported that supplementation with NAD+ precursors (NMN) enhances NAD+ metabolome and ameliorate chemotherapy-induced POI and infertility in mice. Arginine deprivation has been shown to trigger cell cycle arrest, indicating its possible contribution to DOXO-induced DNA damage and cell cycle arrest in ovarian cells. The down-regulation of metabolites related to purine (e.g, Xanthosine-5-phosphate, Hypoxanthine, Inosine) and pyrimidine metabolism (e.g., Uridine, Cytidine, Uracil) in DOXO-treated ovaries indicates compromised nucleotide metabolism, energy metabolism, and cell proliferation or survival. Ongoing studies focus on the identification of cell type-specific metabolic changes and their mechanistic contributions to chemotherapy-induced POI. In summary, our study demonstrates that DOXO alters ovarian metabolome, particularly in nucleotide metabolism, energy balance, and DDR activation. These findings provide novel insights into the metabolic basis of chemotherapy-induced POI, opening avenues for metabolic interventions to preserve young female cancer patients ovarian reserve and fertility. (This work is supported by NIH/NICHD R01HD115810)