Acute pneumocyte injury, poly(ADP-ribose) polymerase activity, and pyridine nucleotide levels after in vitro exposure of murine lung slices to cyclophosphamide.
Cyclophosphamide (CYC) is a metabolically activated, DNA-alkylating, antitumor agent that causes pulmonary fibrosis. BALB/cN (B) mice are sensitive and C57Bl/6N (C) mice are resistant to CYC-induced fibrosis. Pulmonary bioactivation may contribute to strain sensitivity. Therefore, we tested the intrinsic susceptibility of murine lung slices to cell injury by direct exposure to CYC for 2-8 hr. Injury was measured by release of lactate dehydrogenase (LDH). DNA damage activates the nuclear enzyme poly(ADP-ribose) polymerase (PAP, EC 2.4.2.30), causing depletion of its substrate, NAD. NAD can also be decreased by phosphorylation to NADP, as seen with oxidative stress. Depletion of NAD can lead to loss of ATP. Thus, we measured LDH release, PAP activation, NAD, NADP and ATP in slices incubated with or without the PAP-inhibitor, 3-aminobenzamide (3-AB). CYC (0.1 to 1.0 mg/mL for 4-8 hr) caused LDH release in slices from both murine strains, but LDH release was significantly greater in B lung slices than in C slices. After an 8-hr incubation 63.9 +/- 3.7% (mean +/- SEM) of total LDH was released from B lung slices with 1.0 mg CYC/mL, whereas only 45.8 +/- 2.6% was released from C lung slices (P < 0.05). 3-AB reduced LDH release to 44.7 +/- 2.4% in B slices and 28.1 +/- 2.0% in C slices (P < 0.05 vs CYC only). PAP activity in nuclei isolated from CYC-treated B lung slices was increased 2- to 4-fold after 2 hr of incubation with 0.5 and 1.0 mg CYC/mL. PAP activation was delayed and reduced with incubation in 3-AB. PAP was activated 2-fold in nuclei from C slices treated with 0.5 mg CYC/mL for 2 hr. NAD was decreased at 2 and 4 hr in B slices treated with 0.5 and 1.0 mg CYC/mL, and at 4 hr with 0.1 mg CYC/mL. NAD depletion occurred only at 4 hr in the resistant C slices treated with 1.0 mg CYC/mL. CYC increased NADP by a similar extent in B and C lung slices. In B slices, NAD losses were approximately 4 times the increases in NADP. CYC did not decrease ATP in B slices and ATP dropped 25% only after 4 hr in the resistant C slices. We conclude that CYC is directly toxic to lung tissue and observe that strain sensitivity in vitro mirrors the sensitivity to fibrosis in vivo. PAP activation and oxidative stress may contribute to this toxicity.
Hoyt
D G
DG
Department of Pharmacology, University of Pittsburgh, PA 15261.
Lazo
J S
JS
D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D003520:Cyclophosphamide / Q000633:toxicity*; D005260:Female; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008168:Lung / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D051379:Mice; D009243:NAD / Q000032:analysis; D009249:NADP / Q000032:analysis; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011726:Pyridinium Compounds / Q000032:analysis*
D011726:Pyridinium Compounds; D009243:NAD; D009249:NADP; D000255:Adenosine Triphosphate; D003520:Cyclophosphamide; D007770:L-Lactate Dehydrogenase; D011065:Poly(ADP-ribose) Polymerases