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Aileen F Keating - One of the best experts on this subject based on the ideXlab platform.
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ataxia telangiectasia mutated coordinates the ovarian dna repair and atresia initiating response to Phosphoramide Mustard
Biology of Reproduction, 2020Co-Authors: Kendra L Clark, Aileen F KeatingAbstract:Ataxia-telangiectasia-mutated (ATM) protein recognizes and repairs DNA double strand breaks through activation of cell cycle checkpoints and DNA repair proteins. Atm gene mutations increase female reproductive cancer risk. Phosphoramide Mustard (PM) induces ovarian DNA damage and destroys primordial follicles, and pharmacological ATM inhibition prevents PM-induced follicular depletion. Wild-type (WT) C57BL/6 or Atm+/- mice were dosed once intraperitoneally with sesame oil (95%) or PM (25 mg/kg) in the proestrus phase of the estrous cycle and ovaries harvested 3 days thereafter. Atm+/- mice spent ~25% more time in diestrus phase than WT. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) on ovarian protein was performed and bioinformatically analyzed. Relative to WT, Atm+/- mice had 64 and 243 proteins increased or decreased in abundance, respectively. In WT mice, PM increased 162 and decreased 20 proteins. In Atm+/- mice, 173 and 37 proteins were increased and decreased, respectively, by PM. Exportin-2 (XPO2) was localized to granulosa cells of all follicle stages and was 7.2-fold greater in Atm+/- than WT mice. Cytoplasmic FMR1-interacting protein 1 was 6.8-fold lower in Atm+/- mice and was located in the surface epithelium with apparent translocation to the ovarian medulla post-PM exposure. PM induced γH2AX, but fewer γH2AX-positive foci were identified in Atm+/- ovaries. Similarly, cleaved caspase-3 was lower in the Atm+/- PM-treated, relative to WT mice. These findings support ATM involvement in ovarian DNA repair and suggest that ATM functions to regulate ovarian atresia.
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obesity alters Phosphoramide Mustard induced ovarian dna repair in mice
Biology of Reproduction, 2017Co-Authors: Shanthi Ganesan, Jill A Madden, Jackson Nteeba, Aileen F KeatingAbstract:Phosphoramide Mustard (PM) destroys rapidly dividing cells and activates the DNA double strand break marker, γH2AX, and DNA repair in rat granulosa cells and neonatal ovaries. The effects of PM exposure on DNA damage and activation of DNA damage repair in lean and obese female mice were investigated. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice received sesame oil or PM (95%; 25 mg/kg; intraperitoneal injection). Obesity increased (P < 0.05) hepatic and spleen but decreased (P < 0.05) uterine weight. PM exposure reduced (P < 0.05) spleen weight regardless of body composition, however, decreased (P < 0.05) ovarian and hepatic weight were observed in the obese PM-exposed females. PM decreased (P < 0.05) primordial and primary follicle number in lean females. Obesity and PM increased (P < 0.05) γH2AX protein. DNA damage repair genes Prkdc, Parp1, and Rad51 mRNA were unaltered by obesity, however, Atm and Xrcc6 mRNA were increased (P < 0.05) while Brca1 was reduced (P < 0.05). Obesity reduced (P < 0.05) PRKDC, XRCC6 and but increased (P < 0.05) ATM protein. ATM, BRCA1 and RAD51 protein levels were increased (P < 0.05) by PM exposure in both lean and obese mice, while PM-induced increased (P < 0.05) XRCC6 and PARP1 were observed only in lean mice. Thus, PM induces ovarian DNA damage in vivo; obesity alters DNA repair response gene mRNA and protein level; the ovary activates DNA repair proteins in response to PM; but obesity compromises the ovarian PM response.
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progressive obesity alters ovarian insulin phosphatidylinositol 3 kinase and chemical metabolism signaling pathways and potentiates ovotoxicity induced by Phosphoramide Mustard in mice
Biology of Reproduction, 2017Co-Authors: Jackson Nteeba, Jill A Madden, Shanthi Ganesan, M J Dickson, Aileen F KeatingAbstract:Mechanisms underlying obesity-associated reproductive impairment are ill defined. Hyperinsulinemia is a metabolic perturbation often observed in obese subjects. Insulin activates phosphatidylinositol 3-kinase (PI3K) signaling, which regulates ovarian folliculogenesis, steroidogenesis, and xenobiotic metabolism. The impact of progressive obesity on ovarian genes encoding mRNA involved in insulin-mediated PI3K signaling and xenobiotic biotransformation [insulin receptor (Insr), insulin receptor substrate 1 (Irs1), 2 (Irs2), and 3 (Irs3); kit ligand (Kitlg), stem cell growth factor receptor (Kit), protein kinase B (AKT) alpha (Akt1), beta (Akt2), forkhead transcription factor (FOXO) subfamily 1 (Foxo1), and subfamily 3 (Foxo3a), microsomal epoxide hydrolase (Ephx1), cytochrome P450 family 2, subfamily E, polypeptide 1 (Cyp2e1), glutathione S-transferase (GST) class Pi (Gstp1) and class mu 1 (Gstm1)] was determined in normal wild-type nonagouti (a/a; lean) and lethal yellow mice (KK.CG-Ay/J; obese) at 6, 12, 18, or 24 weeks of age. At 6 weeks, ovaries from obese mice had increased (P < 0.05) Insr and Irs3 but decreased (P < 0.05) Kitlg, Foxo1, and Cyp2e1 mRNA levels. Interestingly, at 12 weeks, an increase (P < 0.05) in Kitlg and Kit mRNA, pIRS1Ser302, pAKTThr308, EPHX1, and GSTP1 protein level was observed due to obesity, while Cyp2e1 mRNA and protein were reduced. A Phosphoramide Mustard (PM) challenge increased (P < 0.05) ovarian EPHX1 protein abundance in lean but not obese females. In addition, lung tissue from PM-exposed animals had increased (P < 0.05) EPHX1 protein with no impact of obesity thereon. Taken together, progressive obesity affected ovarian signaling pathways potentially involved in obesity-associated reproductive disorders.
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Phosphoramide Mustard induces autophagy markers and mtor inhibition prevents follicle loss due to Phosphoramide Mustard exposure
Reproductive Toxicology, 2017Co-Authors: Jill A Madden, Porsha Q Thomas, Aileen F KeatingAbstract:Abstract Phosphoramide Mustard (PM) is an ovotoxic metabolite of cyclophosphamide. Postnatal day 4 Fisher 344 rat ovaries were exposed to vehicle control (1% DMSO) or PM (60 μM) ± LY294002 or rapamycin for 2 or 4 d. Transmission election microscopy revealed abnormally large golgi apparatus and electron dense mitochondria in PM-exposed ovaries prior to and at the time of follicle depletion. PM exposure increased (P
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Phosphoramide Mustard induces autophagy markers and mtor inhibition prevents follicle loss due to Phosphoramide Mustard exposure
Reproductive Toxicology, 2017Co-Authors: Jill A Madden, Porsha Q Thomas, Aileen F KeatingAbstract:Phosphoramide Mustard (PM) is an ovotoxic metabolite of cyclophosphamide. Postnatal day 4 Fisher 344 rat ovaries were exposed to vehicle control (1% DMSO) or PM (60μM)±LY294002 or rapamycin for 2 or 4 d. Transmission election microscopy revealed abnormally large golgi apparatus and electron dense mitochondria in PM-exposed ovaries prior to and at the time of follicle depletion. PM exposure increased (P<0.05) mRNA abundance of Bbc3, Cdkn1a, Ctfr, Edn1, Gstp1, Nqo1, Tlr4, Tnfrsfla, Txnrd1 and decreased (P<0.05) Casp1 and Il1b after 4d. PM exposure increased (P<0.1) BECN1 and LAMP, decreased (P<0.1) ABCB1 and did not alter ABCC1 protein. LY294002 did not impact PM-induced ovotoxicity, but decreased ABCC1 and ABCB1 protein. Rapamycin prevented PM-induced follicle loss. These data suggest that the mammalian target of rapamycin, mTOR, may be a gatekeeper of PM-induced follicle loss.
Jos H. Beijnen - One of the best experts on this subject based on the ideXlab platform.
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High exposures to bioactivated cyclophosphamide are related to the occurrence of veno-occlusive disease of the liver following high-dose chemotherapy
British Journal of Cancer, 2006Co-Authors: Milly E. De Jonge, Jos H. BeijnenAbstract:We investigated whether the occurrence of veno-occlusive disease of the liver (VOD) may be associated with individual variations in the pharmacokinetics of high-dose cyclophosphamide. Patients received single or multiple courses of cyclophosphamide (1000 or 1500 mg m^−2 day^−1), thiotepa (80 or 120 mg m^−2 day^−1) and carboplatin (265–400 mg m^−2 day^−1) (CTC) for 4 consecutive days. The area under the plasma concentration–time curves (AUCs) were calculated for cyclophosphamide and its activated metabolites 4-hydroxycyclophosphamide and Phosphoramide Mustard based on multiple blood samples. Possible relationships between the AUCs and the occurrence of VOD were studied. A total of 59 patients (115 courses) were included. Four patients experienced VOD after a second CTC course. The first-course AUC of 4-hydroxycyclophosphamide ( P =0.003) but not of Phosphoramide Mustard ( P =0.101) appeared to be predictive of the occurrence of VOD after multiple courses. High exposures to bioactivated cyclophosphamide may lead to increased organ toxicity.
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population pharmacokinetics of cyclophosphamide and its metabolites 4 hydroxycyclophosphamide 2 dechloroethylcyclophosphamide and Phosphoramide Mustard in a high dose combination with thiotepa and carboplatin
Therapeutic Drug Monitoring, 2005Co-Authors: Milly E. De Jonge, Jos H. BeijnenAbstract:Abstract:The anticancer prodrug cyclophosphamide (CP) is activated by the formation of 4-hydroxycyclophosphamide (4OHCP), which decomposes into Phosphoramide Mustard (PM). This activation pathway is inhibited by thiotepa. CP is inactivated by formation of 2-dechloroethylcyclophosphamide (2DCECP). Th
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Population pharmacokinetics of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide, 2-dechloroethylcyclophosphamide, and Phosphoramide Mustard in a high-dose combination with Thiotepa and Carboplatin.
Therapeutic Drug Monitoring, 2005Co-Authors: Milly E. De Jonge, Sjoerd Rodenhuis, Alwin D. R. Huitema, Jos H. BeijnenAbstract:The anticancer prodrug cyclophosphamide (CP) is activated by the formation of 4-hydroxycyclophosphamide (4OHCP), which decomposes into Phosphoramide Mustard (PM). This activation pathway is inhibited by thiotepa. CP is inactivated by formation of 2-dechloroethylcyclophosphamide (2DCECP). The aim of this study was to develop a population pharmacokinetic model describing the complex pharmacokinetics of CP, 4OHCP, 2DCECP, and PM when CP is administered in a high-dose combination with thiotepa and carboplatin. Patients received a combination of CP (1000-1500 mg/m/d), carboplatin (265-400 mg/m/d), and thiotepa (80-120 mg/m/d) administered in short infusions over 4 days. Twenty blood samples were collected per patient per course. Concentrations of CP, 4OHCP, 2DCECP, PM, thiotepa, and tepa were determined in plasma. Using NONMEM, an integrated population pharmacokinetic model was used to describe the pharmacokinetics of CP, 4OHCP, 2DCECP, and PM, including the already described processes of autoinduction of CP and the interaction with thiotepa. Data were available on 35 patients (70 courses). The pharmacokinetics of CP were described with a 2-compartment model, and those of 4OHCP, 2DCECP, and PM with 1-compartment models. Before onset of autoinduction, it was assumed that CP is eliminated through a noninducible pathway accounting for 20% of total CP clearance, whereas 2 inducible pathways resulted in formation of 4OHCP (75%) and 2DCECP (5%). It was assumed that 4OHCP was fully converted to PM. Induction of CP metabolism was mediated by 2 hypothetical amounts of enzyme whose quantities increased in time in the presence of CP (kenz=0.0223 and 0.0198 hours). Induction resulted in an increased formation of 4OHCP (approximately 50%), PM (approximately 50%), and 2DCECP (approximately 35%) during the 4-day course, and concomitant decreased exposure to CP (approximately 50%). The formation of 2DCECP was not inhibited by thiotepa. Apparent volumes of distribution of CP, PM, and 2DCECP could be estimated being 43.7, 55.5, and 18.5 L, respectively. Exposure to metabolites varied up to 9-fold. The complex population pharmacokinetics of CP, 4OHCP, 2DCECP, and PM in combination with thiotepa and carboplatin has been established and may form the basis for further treatment optimization with this combination
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simple and selective determination of the cyclophosphamide metabolite Phosphoramide Mustard in human plasma using high performance liquid chromatography
Journal of Chromatography B: Biomedical Sciences and Applications, 2000Co-Authors: Alwin D. R. Huitema, Sjoerd Rodenhuis, Jos H. Beijnen, M M Tibben, Thomas Kerbusch, Jantien Kettenesvan Den J BoschAbstract:Abstract A simple and selective assay for the determination of the alkylating cyclophosphamide metabolite Phosphoramide Mustard (PM) in plasma was developed and validated. PM was determined after derivatisation by high-performance liquid chromatography (HPLC) with ultraviolet detection at 276 nm. Sample pre-treatment consisted of derivatisation of PM with diethyldithiocarbamate (DDTC) at 70°C for 10 min, followed by extraction with acetonitrile in the presence of 0.7 M sodium chloride. Phase separation occurred due to the high salt content of the aqueous phase. The HPLC system consisted of a C8 column with acetonitrile–0.025 M potassium phosphate buffer, pH 8.0, (32:68, v/v) as the mobile phase. The entire sample handling procedure, from collection at the clinical ward until analysis in the laboratory, was optimised and validated. Calibration curves were linear from 50 to 10 000 ng/ml. The lower limit of quantification and the limit of detection (using a signal-to-noise ratio of 3) were 50 and 40 ng/ml, respectively, using 500 μl of plasma. Within-day and between-day precisions were below 11% over the entire concentration range and the accuracies were between 100 and 106%. PM was found to be stable at −30°C for at least 10 weeks both in plasma and as a DDTC-derivative in a dry sample. A pharmacokinetic pilot study in two patients receiving 1000 mg/m2 CP in a 1-h infusion demonstrated the applicability of the assay.
Yongying Jiang - One of the best experts on this subject based on the ideXlab platform.
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peptide conjugates of 4 aminocyclophosphamide as prodrugs of Phosphoramide Mustard for selective activation by prostate specific antigen psa
Bioorganic & Medicinal Chemistry, 2013Co-Authors: Yongying JiangAbstract:Abstract In our continued effort to develop prodrugs of Phosphoramide Mustard, conjugates of 4-aminocyclophosphamide (4-NH 2 -CPA) with three PSA-specific peptides were synthesized and evaluated as substrates of PSA. These include conjugates of cis -(2 R ,4 R )-4-NH 2 -CPA with a tetrapeptide Succinyl-Ser-Lys-Leu-Gln-OH, a hexapeptide Succinyl-His-Ser-Ser-Lys-Leu-Gln-OH, and a pentapeptide Glutaryl-Hyp-Ala-Ser-Chg-Gln-OH. These conjugates were cleaved by PSA efficiently and exclusively after the expected glutamine residue to release 4-NH 2 -CPA, the activated prodrug form of Phosphoramide Mustard. The cleavage was most efficient for the pentapeptide conjugate 3 (Glutaryl-Hyp-Ala-Ser-Chg-Gln-NH-CPA), which showed a half-life of 55 min with PSA, followed by the hexapeptide conjugate 2 (Succinyl-His-Ser-Ser-Lys-Leu-Gln-NH-CPA) and the tertrapeptide conjugate 1 (Succinyl-Ser-Lys-Leu-Gln-NH-CPA) with half-lives of 6.5 and 12 h, respectively. These results indicate a potential of the conjugate 3 as an anticancer prodrug of Phosphoramide Mustard for selective PSA activation.
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synthesis and stereochemical preference of peptide 4 aminocyclophosphamide conjugates as potential prodrugs of Phosphoramide Mustard for activation by prostate specific antigen psa
Bioorganic & Medicinal Chemistry Letters, 2009Co-Authors: Yongying Jiang, Robert S DipaolaAbstract:In an effort to develop proteolytically activated prodrugs of Phosphoramide Mustard by prostate-specific antigen (PSA), a series of tetrapeptide (Cbz-Ser-Ser-Phe-Tyr)-conjugated 4-aminocyclophosphamide (4-NH(2)-CPA) isomers were synthesized and evaluated as substrates of PSA. The cleavage of the conjugates by PSA were found to be stereoselective as only the two isomers with 4R-configuration were efficiently cleaved by PSA. The cis-(2R,4R)-isomer was the best substrate of PSA with a half-life of 12min. LC/MS analysis of the incubation solution of this isomer with PSA suggests that 4-NH(2)-CPA is released upon proteolysis and quickly degrades to cytotoxic Phosphoramide Mustard. These results clarified the stereochemical requirements of PSA on the peptide conjugates of 4-NH(2)-CPA and demonstrated the potential of these conjugates as potential PSA-activated prodrugs targeting prostate cancer.
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n 2 2 dimethyl 2 2 nitrophenyl acetyl 4 aminocyclophosphamide as a potential bioreductively activated prodrug of Phosphoramide Mustard
Bioorganic & Medicinal Chemistry Letters, 2008Co-Authors: Yongying JiangAbstract:N-(2,2-Dimethyl-2-(2-nitrophenyl)acetyl)-4-aminocyclophosphamide isomers (DMNA-NH-CPA, 4) were synthesized stereospecifically from Boc-L-Hse(OBn)-OH and the degradation of the corresponding reduced amine 5a was investigated by UV/vis spectroscopy and LC/MS. The rate of cyclization of 5a was found to increase with decreasing pH, with half-lives ranging from 3.2 to 54 min at pH 4-7.4, suggesting that the cyclization is catalyzed by the hydronium ions. LC/MS analysis of the degradation products of 5a indicates that 4-aminocyclophosphamide is rapidly released from 4 upon reductive activation under acidic conditions and further decomposes into the cytotoxic Phosphoramide Mustard. These results validated 4-aminocyclophosphamide as a prodrug form of Phosphoramide Mustard and suggest that compound 4 can potentially be used as a prodrug of Phosphoramide Mustard for bioreductive activation.
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Nitroaryl Phosphoramides as novel prodrugs for E. coli nitroreductase activation in enzyme prodrug therapy.
Journal of Medicinal Chemistry, 2003Co-Authors: Yongying Jiang, Jiye Han, Patrick Browne, Peter F. Searle, Paul R. Race, Richard J. Knox, Eva I. HydeAbstract:Cyclic and acyclic nitroaryl Phosphoramide Mustard analogues were activated by E. coli nitroreductase, an enzyme explored in GDEPT. The more active acyclic 4-nitrobenzyl Phosphoramide Mustard (7) showed 167 500x selective cytotoxicity toward nitroreductase-expressing V79 cells with an IC(50) as low as 0.4 nM. This is about 100x more active and 27x more selective than CB1954 (1). The superior activity was attributed to its better substrate activity (k(cat)/K(m) 19x better than 1) and/or excellent cytotoxicity of Phosphoramide Mustard released.
Milly E. De Jonge - One of the best experts on this subject based on the ideXlab platform.
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High exposures to bioactivated cyclophosphamide are related to the occurrence of veno-occlusive disease of the liver following high-dose chemotherapy
British Journal of Cancer, 2006Co-Authors: Milly E. De Jonge, Jos H. BeijnenAbstract:We investigated whether the occurrence of veno-occlusive disease of the liver (VOD) may be associated with individual variations in the pharmacokinetics of high-dose cyclophosphamide. Patients received single or multiple courses of cyclophosphamide (1000 or 1500 mg m^−2 day^−1), thiotepa (80 or 120 mg m^−2 day^−1) and carboplatin (265–400 mg m^−2 day^−1) (CTC) for 4 consecutive days. The area under the plasma concentration–time curves (AUCs) were calculated for cyclophosphamide and its activated metabolites 4-hydroxycyclophosphamide and Phosphoramide Mustard based on multiple blood samples. Possible relationships between the AUCs and the occurrence of VOD were studied. A total of 59 patients (115 courses) were included. Four patients experienced VOD after a second CTC course. The first-course AUC of 4-hydroxycyclophosphamide ( P =0.003) but not of Phosphoramide Mustard ( P =0.101) appeared to be predictive of the occurrence of VOD after multiple courses. High exposures to bioactivated cyclophosphamide may lead to increased organ toxicity.
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population pharmacokinetics of cyclophosphamide and its metabolites 4 hydroxycyclophosphamide 2 dechloroethylcyclophosphamide and Phosphoramide Mustard in a high dose combination with thiotepa and carboplatin
Therapeutic Drug Monitoring, 2005Co-Authors: Milly E. De Jonge, Jos H. BeijnenAbstract:Abstract:The anticancer prodrug cyclophosphamide (CP) is activated by the formation of 4-hydroxycyclophosphamide (4OHCP), which decomposes into Phosphoramide Mustard (PM). This activation pathway is inhibited by thiotepa. CP is inactivated by formation of 2-dechloroethylcyclophosphamide (2DCECP). Th
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Population pharmacokinetics of cyclophosphamide and its metabolites 4-hydroxycyclophosphamide, 2-dechloroethylcyclophosphamide, and Phosphoramide Mustard in a high-dose combination with Thiotepa and Carboplatin.
Therapeutic Drug Monitoring, 2005Co-Authors: Milly E. De Jonge, Sjoerd Rodenhuis, Alwin D. R. Huitema, Jos H. BeijnenAbstract:The anticancer prodrug cyclophosphamide (CP) is activated by the formation of 4-hydroxycyclophosphamide (4OHCP), which decomposes into Phosphoramide Mustard (PM). This activation pathway is inhibited by thiotepa. CP is inactivated by formation of 2-dechloroethylcyclophosphamide (2DCECP). The aim of this study was to develop a population pharmacokinetic model describing the complex pharmacokinetics of CP, 4OHCP, 2DCECP, and PM when CP is administered in a high-dose combination with thiotepa and carboplatin. Patients received a combination of CP (1000-1500 mg/m/d), carboplatin (265-400 mg/m/d), and thiotepa (80-120 mg/m/d) administered in short infusions over 4 days. Twenty blood samples were collected per patient per course. Concentrations of CP, 4OHCP, 2DCECP, PM, thiotepa, and tepa were determined in plasma. Using NONMEM, an integrated population pharmacokinetic model was used to describe the pharmacokinetics of CP, 4OHCP, 2DCECP, and PM, including the already described processes of autoinduction of CP and the interaction with thiotepa. Data were available on 35 patients (70 courses). The pharmacokinetics of CP were described with a 2-compartment model, and those of 4OHCP, 2DCECP, and PM with 1-compartment models. Before onset of autoinduction, it was assumed that CP is eliminated through a noninducible pathway accounting for 20% of total CP clearance, whereas 2 inducible pathways resulted in formation of 4OHCP (75%) and 2DCECP (5%). It was assumed that 4OHCP was fully converted to PM. Induction of CP metabolism was mediated by 2 hypothetical amounts of enzyme whose quantities increased in time in the presence of CP (kenz=0.0223 and 0.0198 hours). Induction resulted in an increased formation of 4OHCP (approximately 50%), PM (approximately 50%), and 2DCECP (approximately 35%) during the 4-day course, and concomitant decreased exposure to CP (approximately 50%). The formation of 2DCECP was not inhibited by thiotepa. Apparent volumes of distribution of CP, PM, and 2DCECP could be estimated being 43.7, 55.5, and 18.5 L, respectively. Exposure to metabolites varied up to 9-fold. The complex population pharmacokinetics of CP, 4OHCP, 2DCECP, and PM in combination with thiotepa and carboplatin has been established and may form the basis for further treatment optimization with this combination
Shanthi Ganesan - One of the best experts on this subject based on the ideXlab platform.
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obesity alters Phosphoramide Mustard induced ovarian dna repair in mice
Biology of Reproduction, 2017Co-Authors: Shanthi Ganesan, Jill A Madden, Jackson Nteeba, Aileen F KeatingAbstract:Phosphoramide Mustard (PM) destroys rapidly dividing cells and activates the DNA double strand break marker, γH2AX, and DNA repair in rat granulosa cells and neonatal ovaries. The effects of PM exposure on DNA damage and activation of DNA damage repair in lean and obese female mice were investigated. Wild type (lean) non agouti (a/a) and KK.Cg-Ay/J heterozygote (obese) mice received sesame oil or PM (95%; 25 mg/kg; intraperitoneal injection). Obesity increased (P < 0.05) hepatic and spleen but decreased (P < 0.05) uterine weight. PM exposure reduced (P < 0.05) spleen weight regardless of body composition, however, decreased (P < 0.05) ovarian and hepatic weight were observed in the obese PM-exposed females. PM decreased (P < 0.05) primordial and primary follicle number in lean females. Obesity and PM increased (P < 0.05) γH2AX protein. DNA damage repair genes Prkdc, Parp1, and Rad51 mRNA were unaltered by obesity, however, Atm and Xrcc6 mRNA were increased (P < 0.05) while Brca1 was reduced (P < 0.05). Obesity reduced (P < 0.05) PRKDC, XRCC6 and but increased (P < 0.05) ATM protein. ATM, BRCA1 and RAD51 protein levels were increased (P < 0.05) by PM exposure in both lean and obese mice, while PM-induced increased (P < 0.05) XRCC6 and PARP1 were observed only in lean mice. Thus, PM induces ovarian DNA damage in vivo; obesity alters DNA repair response gene mRNA and protein level; the ovary activates DNA repair proteins in response to PM; but obesity compromises the ovarian PM response.
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progressive obesity alters ovarian insulin phosphatidylinositol 3 kinase and chemical metabolism signaling pathways and potentiates ovotoxicity induced by Phosphoramide Mustard in mice
Biology of Reproduction, 2017Co-Authors: Jackson Nteeba, Jill A Madden, Shanthi Ganesan, M J Dickson, Aileen F KeatingAbstract:Mechanisms underlying obesity-associated reproductive impairment are ill defined. Hyperinsulinemia is a metabolic perturbation often observed in obese subjects. Insulin activates phosphatidylinositol 3-kinase (PI3K) signaling, which regulates ovarian folliculogenesis, steroidogenesis, and xenobiotic metabolism. The impact of progressive obesity on ovarian genes encoding mRNA involved in insulin-mediated PI3K signaling and xenobiotic biotransformation [insulin receptor (Insr), insulin receptor substrate 1 (Irs1), 2 (Irs2), and 3 (Irs3); kit ligand (Kitlg), stem cell growth factor receptor (Kit), protein kinase B (AKT) alpha (Akt1), beta (Akt2), forkhead transcription factor (FOXO) subfamily 1 (Foxo1), and subfamily 3 (Foxo3a), microsomal epoxide hydrolase (Ephx1), cytochrome P450 family 2, subfamily E, polypeptide 1 (Cyp2e1), glutathione S-transferase (GST) class Pi (Gstp1) and class mu 1 (Gstm1)] was determined in normal wild-type nonagouti (a/a; lean) and lethal yellow mice (KK.CG-Ay/J; obese) at 6, 12, 18, or 24 weeks of age. At 6 weeks, ovaries from obese mice had increased (P < 0.05) Insr and Irs3 but decreased (P < 0.05) Kitlg, Foxo1, and Cyp2e1 mRNA levels. Interestingly, at 12 weeks, an increase (P < 0.05) in Kitlg and Kit mRNA, pIRS1Ser302, pAKTThr308, EPHX1, and GSTP1 protein level was observed due to obesity, while Cyp2e1 mRNA and protein were reduced. A Phosphoramide Mustard (PM) challenge increased (P < 0.05) ovarian EPHX1 protein abundance in lean but not obese females. In addition, lung tissue from PM-exposed animals had increased (P < 0.05) EPHX1 protein with no impact of obesity thereon. Taken together, progressive obesity affected ovarian signaling pathways potentially involved in obesity-associated reproductive disorders.
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the ovarian dna damage repair response is induced prior to Phosphoramide Mustard induced follicle depletion and ataxia telangiectasia mutated inhibition prevents pm induced follicle depletion
Toxicology and Applied Pharmacology, 2016Co-Authors: Shanthi Ganesan, Aileen F KeatingAbstract:Phosphoramide Mustard (PM) is an ovotoxic metabolite of cyclophosphamide and destroys primordial and primary follicles potentially by DNA damage induction. The temporal pattern by which PM induces DNA damage and initiation of the ovarian response to DNA damage has not yet been well characterized. This study investigated DNA damage initiation, the DNA repair response, as well as induction of follicular demise using a neonatal rat ovarian culture system. Additionally, to delineate specific mechanisms involved in the ovarian response to PM exposure, utility was made of PKC delta (PKCδ) deficient mice as well as an ATM inhibitor (KU 55933; AI). Fisher 344 PND4 rat ovaries were cultured for 12, 24, 48 or 96h in medium containing DMSO ±60μM PM or KU 55933 (48h; 10nM). PM-induced activation of DNA damage repair genes was observed as early as 12h post-exposure. ATM, PARP1, E2F7, P73 and CASP3 abundance were increased but RAD51 and BCL2 protein decreased after 96h of PM exposure. PKCδ deficiency reduced numbers of all follicular stages, but did not have an additive impact on PM-induced ovotoxicity. ATM inhibition protected all follicle stages from PM-induced depletion. In conclusion, the ovarian DNA damage repair response is active post-PM exposure, supporting that DNA damage contributes to PM-induced ovotoxicity.
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Phosphoramide Mustard exposure induces dna adduct formation and the dna damage repair response in rat ovarian granulosa cells
Toxicology and Applied Pharmacology, 2015Co-Authors: Shanthi Ganesan, Aileen F KeatingAbstract: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 6 μM) for 24 or 48 h. Cell viability was reduced (P < 0.05) after 48 h of exposure to 3 or 6 μM PM. The NOR-G-OH DNA adduct was detected after 24 h of 6 μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48 h 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.