title: Phosphoramide mustard exposure induces DNA adduct formation and the DNA damage repair response in rat ovarian granulosa cells.
abstract: Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6μM) for 24 or 48h. Cell viability was reduced (P<0.05) after 48h of exposure to 3 or 6μM PM. The NOR-G-OH DNA adduct was detected after 24h of 6μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response.
authors.affiliation: Department of Animal Science, Iowa State University, Ames, IA 50011, USA. Electronic address: shanthig@iastate.edu.
authors.forename: Shanthi
authors.initials: S
authors.lastname: Ganesan
authors.affiliation: Department of Animal Science, Iowa State University, Ames, IA 50011, USA. Electronic address: akeating@iastate.edu.
authors.forename: Aileen F
authors.initials: AF
authors.lastname: Keating
mesh_terms: D000818:Animals; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000633:toxicity*; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism; D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D018736:DNA Adducts / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D006107:Granulosa Cells / Q000187:drug effects* / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D000072200:Ku Autoantigen; D010750:Phosphoproteins / Q000378:metabolism; D010752:Phosphoramide Mustards / Q000633:toxicity*; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D051135:Rad51 Recombinase / Q000235:genetics / Q000378:metabolism; D051381:Rats
chemical_list: D034961:Antigens, Nuclear; D000970:Antineoplastic Agents; D019313:BRCA1 Protein; D018736:DNA Adducts; D004268:DNA-Binding Proteins; D006657:Histones; D010750:Phosphoproteins; D010752:Phosphoramide Mustards; C577575:gamma-H2AX protein, rat; C030090:phosphoramide mustard; C499322:Parp1 protein, rat; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; D051135:Rad51 Recombinase; C495155:Rad51 protein, rat; C000606511:Xrcc6 protein, rat; D000072200:Ku Autoantigen
keywords: Cyclophosphamide; DNA adduct; DNA damage; DNA repair; Granulosa cells; Phosphoramide mustard