Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Aug 12;8(8):e71592.
doi: 10.1371/journal.pone.0071592. eCollection 2013.

Supplementation with fish oil and genistein, individually or in combination, protects bone against the adverse effects of methotrexate chemotherapy in rats

Rethi Raghu Nadhanan  1 , Jayne Skinner, Rosa Chung, Yu-Wen Su, Peter R Howe, Cory J Xian
Affiliations

Supplementation with fish oil and genistein, individually or in combination, protects bone against the adverse effects of methotrexate chemotherapy in rats

Rethi Raghu Nadhanan et al. PLoS One. .

Abstract

Cancer chemotherapy has been shown to induce long-term skeletal side effects such as osteoporosis and fractures; however, there are no preventative treatments. This study investigated the damaging effects of anti-metabolite methotrexate (MTX) subcutaneous injections (0.75 mg/kg BW) for five days and the potential protective benefits of daily oral gavage of fish oil at 0.5 mL/100 g BW (containing 375 mg of n-3 PUFA/100 g BW), genistein (2 mg/100 g BW), or their combination in young adult rats. MTX treatment alone significantly reduced primary spongiosa height and secondary spongiosa trabecular bone volume. Bone marrow stromal cells from the treated rats showed a significant reduction in osteogenic differentiation but an increase in adipogenesis ex vivo. Consistently, stromal cells had significantly higher mRNA levels of adipogenesis-related proliferator activator activated receptor-γ (PPAR-γ) and fatty acid binding protein (FABP4). MTX significantly increased the numbers of bone-resorbing osteoclasts and marrow osteoclast precursor cell pool while significantly enhancing the mRNA expression of receptor activator for nuclear factor kappa B ligand (RANKL), the RANKL/osteoprotegerin (OPG) ratio, interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in the bone. Supplementary treatment with fish oil and/or genistein significantly preserved trabecular bone volume and osteogenesis but suppressed MTX-induced adipogenesis and increases in osteoclast numbers and pro-osteoclastogenic cytokine expression. Thus, Fish oil and/or genistein supplementation during MTX treatment enabled not only preservation of osteogenic differentiation, osteoblast number and bone volume, but also prevention of MTX treatment-induced increases in bone marrow adiposity, osteoclastogenic cytokine expression and osteoclast formation, and thus bone loss.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Effects of MTX with or without fish oil (FO) and/or genistein (Gen) supplementation on growth plate, primary and secondary spongiosa.
Paraffin sections of the tibial metaphysis region (PS =  Primary spongiosa, SS =  Secondary spongiosa, which are separated by a dashed line) of (A) a normal rat, (B) a MTX+H2O treated rat showing reduced height of primary spongiosa and. metaphyseal bone volume, (C) a MTX+FO treated rat, and (D) a MTX+Gen treated rat. (E) Growth plate total height (µm). (F) Primary spongiosa height (µm). (G) Secondary spongiosa BV/TV (%). (H) Secondary spongiosa osteoblast/mm2 trabecular bone area. Values are means ± SEM; n = 7–8 for all groups. Labelled means without a common letter differ (P<0.05).
Figure 2
Figure 2. Effects of MTX alone or with supplementary treatment with (FO) and/or genistein (Gen) on osteogenic differentiation potential of bone marrow stromal cells isolated from treated rats.
Images of a culture well showing bone marrow stromal cell-derived CFU-f colonies stained positive for alkaline phosphatase (ALP, arrows) of (A) a control rat and (B) a MTX alone treated rat on day 9 post the first MTX injection. (C) Treatment effects on size of osteoprogenitor cell pool in bone marrow. Mineralizing colonies stained positive by Alizarin Red (arrows) of (D) a control rat and (E) a MTX alone treated rat. (F) Ex vivo mineralization assay with bone marrow cells isolated from rats. RT-PCR relative mRNA expression of (G) Runx2 and of (H) bone matrix protein osteocalcin (OCN) assessed in bone marrow stromal cells of treated rats (relative to Cyclophilin-A). Labelled means without a common letter differ (P<0.05).
Figure 3
Figure 3. Effects of MTX with or without (FO) and/or genistein (Gen) supplementation on bone marrow adiposity and adipogenesis potential ex vivo.
H&E-stained sections of tibial lower secondary spongiosa in a (A) control rat and (B) a MTX alone treated rat. (C) Adipocyte numbers on bone histology sections. (D) Nile Red-stained images of cultures showing adipocyte formation in an ex vivo adipogenesis assay with bone marrow stromal cells of a control rat and (E) a MTX+H2O treated rat. (F) Quantification of Nile Red+ colonies in an ex vivo adipogenesis assay from bone marrow cells of treated rats. RT-PCR relative gene expression analysis of adipogenesis related genes (G) PPARγ and (H) FABP4 assessed in the isolated bone marrow stromal cells. Labelled means without a common letter differ (P<0.05).
Figure 4
Figure 4. Effects of MTX with or without (FO) and/or genistein (Gen) supplementation on osteoclastogenesis potential.
Images of TRAP-stained tibial metaphysis (arrows pointing multinucleated TRAP-positive osteoclasts) of (A) a control rat and (B) a MTX alone treated rat on day 9 post the first MTX injection showing more osteoclasts present. (C) Average osteoclast numbers at tibial primary and secondary spongiosa. Images of TRAP positively-stained cells formed (arrows pointing multinucleated TRAP-positive osteoclast-like cells) in an ex vivo osteoclastogenesis assay of (D) a control rat and (E) a MTX alone treated rat on day 9 post the first MTX injection showing more osteoclasts formed. (F) Ex vivo osteoclast formation from bone marrow cells isolated from treated rats. Labelled means without a common letter differ (P<0.05).
Figure 5
Figure 5. Effects of MTX with or without (FO) and/or genistein (Gen) supplementation on expression of osteoclastogenesis-regulatory or related genes.
Levels of mRNA expression in metaphysis bones of treated rats as quantitated by real time RT-PCR: (A) RANKL/OPG ratio, (B) TNF-α, and (C) IL-6. (D) Levels (pg/mL) of circulating IL-6 protein in plasma samples of treated rats as measured by multiplex cytokine assay. Labelled means without a common letter differ (P<0.05).
Figure 6
Figure 6. Effects of MTX with or without (FO) and/or genistein (Gen) supplementation on expression of anti-inflammatory cytokines IL-4 and IL-10.
Levels of mRNA expression in metaphysis bones of treated rats as quantitated by real time RT-PCR: (A) IL-4 and (B) IL-10. Protein levels (pg/mL) of circulating (C) IL-4 and (D) IL-10 in plasma samples of treated rats as measured by multiplex cytokine assay. Labelled means without a common letter differ (P<0.05).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Fan C, Cool JC, Scherer MA, Foster BK, Shandala T, et al. (2009) Damaging effects of chronic low-dose methotrexate usage on primary bone formation in young rats and potential protective effects of folinic acid supplementary treatment. Bone 44: 61–70. - PubMed
    1. Xian CJ, Cool JC, Pyragius T, Foster BK (2006) Damage and recovery of the bone growth mechanism in young rats following 5-fluorouracil acute chemotherapy. J Cell Biochem 99: 1688–1704. - PubMed
    1. Xian CJ, Cool JC, Scherer MA, Macsai CE, Fan C, et al. (2007) Cellular mechanisms for methotrexate chemotherapy-induced bone growth defects. Bone 41: 842–850. - PubMed
    1. Xian CJ, Cool JC, van Gangelen J, Foster BK, Howarth GS (2007) Effects of etoposide and cyclophosphamide acute chemotherapy on growth plate and metaphyseal bone in rats. Cancer Biol Ther 6: 170–177. - PubMed
    1. Atkinson SA, Fraher L, Gundberg CM, Andrew M, Pai M, et al. (1989) Mineral homeostasis and bone mass in children treated for acute lymphoblastic leukemia. J Pediatr 114: 793–800. - PubMed

Publication types

MeSH terms

Grants and funding

This project was funded in part by project grants from Channel-7 Children’s Research Foundation of South Australia and National Health and Medical Research Council (NHMRC) Australia. RRN is a recipient of University of South Australia PhD scholarship. CJX is a senior fellow of NHMRC Australia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.