Endocrine active nutrients explored in human bone cell cultures Dr. - - PowerPoint PPT Presentation

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Endocrine active nutrients explored in human bone cell cultures Dr. - - PowerPoint PPT Presentation

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Endocrine active nutrients explored in human bone cell cultures Dr. Igor Bendik Human Nutrition and Health, NIC-RD/HN, DSM Nutritional Products (DNP) December 17, 2012 Overview DSM Family DSM Nutritional Products Endocrine active

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Endocrine active nutrients explored in human bone cell cultures

  • Dr. Igor Bendik

Human Nutrition and Health, NIC-RD/HN, DSM Nutritional Products (DNP) December 17, 2012

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Overview

DSM Family – DSM Nutritional Products Endocrine active substances – in Nutrition Bone Cell Culture Models Endocrine Disruptor Consideration Conclusion

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Page Latin America: 15 locations 1,000 employees North America: 33 locations 4,000 employees Europe: 64 locations 13,000 employees China: 31 locations 3,000 employees India: 8 locations 700 employees Page 2

2011 World total: 200+ locations, 22,000 employees

DSMs global presence

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  • Net sales

€ 3,370m

  • EBITDA

€ 735m

  • EBITDA margin

21.8%

  • R&D

€ 200m

  • Workforce

8,329

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DSM Nutrition at a glance 2011

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Endocrine active substances in nutrition

In Foods or supplements

  • Vitamins
  • Carotenoids
  • Nutraceuticals
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Nutraceuticals vs Endocrine disruptors

Nutraceutical

  • Natural substances from foods

that provide physiological health benefits or provide protection against chronic diseases

  • Nutritional origin
  • Molecular effects supportive

Endocrine disruptor

  • Exogenous substances altering

function(s) of the endocrine system to cause adverse health effects in an intact organism, or its progeny, or (sub)populations

  • Non-nutritional origin
  • Molecular effects disruptive
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Animal welfare – Ethical commitments

Reduction of the number of animals tested Refinement of the methodology Replacement of the animal model

RRR

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Example: Bone biology Endocrine active substances (EASs)

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Source: http://www.bonekey-ibms.org/

  • Bone

remodeling is a balanced coupling of bone formation and resorption

  • Two different

cell types play a role: Osteoblasts and Osteoclasts

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Human primary bone cultures In vitro bone models (osteoblasts)

  • 1. Mesenchymal Stem Cells (BMSC)

isolated from bone marrow, differentiation through Osteostimulation

  • 2. Primary Osteoblast Cells (pOB)

collagenase-digest of human trabecular bone

  • 3. Primary Periost Cells

collagenase-digest of periostal layer

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Human primary bone culture Primary osteoblasts

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pOB cell culture model For reproducible results

  • Characterize pOB batch using

gene expression fingerprint (profiling)

  • Perform analysis with 4-6

passages after the harvest of the primary cell culture

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pOB Affymetrix GeneChip Array Analysis

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Functional expression analysis in primary human osteoblasts

Genistein and the endogenous hormone 17beta- estradiol have identical gene regulation pattern on selected bone marker genes

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DNP Strategy on nutraceutical safety Toxicology and ADME

  • Literature assessment
  • In silico assessment

– Toxicitiy alerts (DEREK Software) – Metabolism (METEOR Software)

  • Screens

– Genotoxicity (in vitro micronucleus test) – Additional investigations as necessary (e.g nuclear receptor test battery)

  • Regulatory safety package
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Non-mandatory DNP screening battery for endocrine disruptors

Case-by-case decision depending on: I. Structural alert II. Systemic exposure

  • III. Results of available in vivo toxicological information
  • IV. Regulatory environment
  • V. Customer expectation
  • In vitro (with parent and possibly metabolites)

– ER transactivation assay – AR transactivation assay – Steroidogenesis assay – Aromatase assay

  • In vivo

– uterotropic and Hershberger assay (if positive in vitro)

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Conclusions

Primary bone cell cultures

  • Different human primary bone cell culture models available for bone

biology (for efficacy testing)

  • Certain vitamins, nutraceuticals function through nuclear receptors

also in bone tissue (along the endocrine axis)

  • Limitation of primary cell culture model for biological questions:

– No systemic approach – Knowledge on pathways important – Comparative analysis using controls only: targeted approach

Safety assessment of endocrine disruption (ED) in bone cells

  • ED analysis using primary bone cells requests mechanistic knowledge
  • n detrimental homeostasis disruption

– Whole transcriptome analysis possible (Affymetrix or Illumina), but will require validation

  • DNP Safety evaluation approach, OECD recommendation
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Thank you for your attention

igor.bendik@dsm.com

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