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 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.

Characterization of Nitrogen Mustard Formamidopyrimidine Adduct Formation of Bis(2-chloroethyl)ethylamine with Calf Thymus DNA and a Human Mammary Cancer Cell Line

“…The chemistry and reactivity of nitrogen 8-10, 18, 20, 24, 47-56 and sulfur 44, 57-64 mustards with nucleosides and DNA in vitro is well studied. There are also numerous studies that examine the mustard derived DNA adducts from treated cell culture and animals, 46,48,50,54,56,59,64,65 including white blood cells of patients on chemotherapeutic regimens that include a nitrogen mustard.…”

“…53, 57 A variety of methods have been used to identify these adducts including 32 P-postlabeling, 47, 58 co-chromatography by HPLC with authentic standards, 8, 9, 24, 48, 57 and mass spectrometry. 18, 20, 49-52, 56, 59, 64 Recently, tandem mass spectrometry with stable isotope dilution has been employed to identify and quantitate some of these DNA adducts. 53, 54, 61-63 Collectively, the studies indicate that these electrophiles react predominantly at the N7 atom of dG, although N3-dA modification can also be significant.…”

Inhibitors of apoptosis protect the ovarian reserve from cyclophosphamide

“…CPA is activated by CYP2B and CYP3A to produce 4-hydroxycyclophosphamide (4-OH-CPA), which can be interconverted to aldophosphamide (AP). AP can be metabolized to phosphoramide mustard (PM) (Ludeman 1999), which causes rapid destruction of proliferating cells by inducing apoptotic pathways through DNA damage (Petrillo et al 2011, Madden et al 2014, Ganesan & Keating 2015. 4-HC is a congener of CPA that was first manufactured in the 1970s (Van der Steen et al 1973, Takamizawa et al 1975 and can be easily converted into the key metabolite 4-OH-CPA without an enzymatic reaction.…”

Phase III biotransformation enzyme involvement in the ovarian response to ovotoxic environmental exposures

“…Cells were treated with vehicle control (1% DMSO) or PM (6 µM) for 48 h for protein isolation. This PM concentration and time point was previously demonstrated to cause approximately 30% cell death [63].…”

“…Activin produced by GC amplifies FSH secretion by boosting formation of GnRH receptors [62,63]. However, activin is inhibited by inhibin and follistatin.…”