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Intermittent PTH1–34 Causes DNA and Chromosome Breaks in Osteoblastic and Nonosteoblastic Cells

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Abstract

Toxicological studies have demonstrated that intermittent PTH1–34 treatment is associated with an increased incidence of osteosarcoma in Fischer 344 rats. Comet and micronucleus (MN) tests, standard methods to evaluate genotoxic potential of drugs, were used to detect DNA and chromosome breaks, respectively, after PTH1–34 treatment. MC3T3 cells, primary osteoblast calvarial cells, and human osteoblasts were treated with PTH1–34 (50 and 100 nM) for 6 h/day for 21 days to mimic intermittent administration. Genotoxic assays were performed at 6 h and 7, 14, and 21 days. Osteoblasts extracted from bone marrow of mice treated with daily subcutaneous PTH1–34 injections (20 and 40 μg/kg) for 10 weeks as well as Hep-2, HeLa, and Hep-G2 cells were also tested. We observed a significant increase in DNA lesions and MN prevalence in human and murine osteoblasts treated with PTH1–34 compared to controls (P < 0.01). The effect observed in vitro and confirmed in vivo was time- and dose-dependent. For nonosteoblastic Hep-2 and HeLa cells we observed increased DNA damage and MN prevalence only later in the course of the protocol, after 21 days of treatment (P < 0.01). In Hep-G2 cells intermittent PTH1–34 did not induce DNA damage or chromosome breaks. Our results demonstrated that intermittent PTH increases DNA and chromosome breaks in osteoblasts. This genotoxic effect is attenuated in nonosteoblastic cells, and the ability to induce DNA damage is lost in cells with detoxification properties (HepG2 cells) tested in vitro.

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Acknowledgments

The authors are indebted to Dr. Rosa Maria Rodrigues Pereira, Rheumatology Division, São Paulo University, for providing MC3T3 cells and to Dr. Aluisio Barbosa de Carvalho, Nephrology Division, UNIFESP, for the histomorphometric analyses. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Brazil. E. C. A. O. received a doctoral scholarship from the CAPES Foundation, Ministry of Education, Brazil.

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  1. Rheumatology Division, Universidade Federal de São Paulo, Escola Paulista de Medicina, São Paulo, Brazil

    Elisângela Cláudia Alves de Oliveira, Vera Lúcia Szejnfeld, Neusa Pereira da Silva, Luís Eduardo Coelho Andrade & Charlles Heldan de Moura Castro

  2. Disciplina de Reumatologia, Rua Botucatu, 740, 3° andar, Vila Clementino, 04023-900, São Paulo, Brazil

    Charlles Heldan de Moura Castro

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  1. Elisângela Cláudia Alves de Oliveira
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  3. Neusa Pereira da Silva
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Correspondence to Charlles Heldan de Moura Castro.

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223_2010_9396_MOESM1_ESM.jpg

Supplemental Fig. 1 Intermittent PTH1–34 induces DNA breaks in MC3T3 cells. Representative sample image of in vitro comet assay. MC3T3 cells grown in αMEM supplemented with 10% FCS and antibiotics were treated 6 h/day for 21 days with PTH1–34 at 50 and 100 nM. a MC3T3 cells treated intermittently with PTH1–34 at 100 nM for 14 days. b Negative control (JPEG 386 kb)

223_2010_9396_MOESM2_ESM.jpg

Supplemental Fig. 2 Intermittent PTH1–34 induces DNA breaks in human osteoblasts. Representative sample image of in vitro assay. Human osteoblasts grown in αMEM supplemented with 10% FCS and antibiotics were treated 6 h/day for 21 days with PTH1–34 at 50 and 100 nM. a Human osteoblasts treated intermittently with PTH1–34 at 50 nM for 21 days. b Negative control (JPEG 366 kb)

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Alves de Oliveira, E.C., Szejnfeld, V.L., Pereira da Silva, N. et al. Intermittent PTH1–34 Causes DNA and Chromosome Breaks in Osteoblastic and Nonosteoblastic Cells. Calcif Tissue Int 87, 424–436 (2010). https://doi.org/10.1007/s00223-010-9396-6

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  • DOI: https://doi.org/10.1007/s00223-010-9396-6

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