Estrogenic bioactivity due to phytoestrogens; genistein and daidzein in soya based weaning flours (Case of Nakuru)

Transcription

1 Estrogenic bioactivity due to phytoestrogens; genistein and daidzein in soya based weaning flours (Case of Nakuru) Presenter: Dr. P. M. Ngoda 24/08/2016 1

2 Background Soya based weaning foods have been used extensively in Kenya due to the following reasons the high protein content allergy to milk lactose intolerance and Easy to make (Add flour blend in some cold water and mix to a paste, add it into boiling water and keep stirring until it boils and does not sink to the bottom of the pan. Leave to boil until cooked.) 24/08/2016 2

3 Effects of soya presumed health benefits of soya bean (Glycine max. L) : reduced cancer risk, cholesterol homeostasis, good for bones and cardiovascular health Adverse effects, Cancer, prepubertal gynocomastia, cryptochidism, hypospadias, reduced fertility The porridge flour blends Soya bean is a legume not used alone to make porridges but in combination with other flours. 24/08/2016 3

4 Common ingredients mixed with soy Millet Peanuts sorghum Maize Soya beans Beans Sweet potatoes Pigeon peas Figure 1 Cassava 4

5 Phytoestrogens may act through the hormone receptors due to their resemblance to 17-β estradiol Figure 2 Phytoestrogens compared to 17 β-estradiol 24/08/2016 5

6 Why baby food...? Infants and young children are more vulnerable because Limited in their diet they feed frequently on the foods under investigation Have less body weight and hence can get high plasma levels of the given compounds Many of their biochemical systems not fully developed and hence susceptible to endocrine disruption; have developmental critical windows of sensitivity to the toxic effects of EDCs 24/08/2016 6

7 Risk assessment Food estimation methods Food Records Food frequency Questionnaires Estrogenic and Androgenic potential Reporter Gene Assays using MMV-Luc and TARM-Luc cell lines 24/08/2016 7

8 Aim To assess the exposure levels of genistein and daidzein to the babies being fed soya based gruels estrogenic equivalents. Exposure assessment is a measure of the amount of hazard to which a person or organism is exposed, or the likelihood of being exposed and is a means of assessing risk, where: Risk = hazard x exposure (Thomson, 2010). 24/08/2016 8

9 Assumptions: Mean values for genistein per gram of soy flour 1750µg/g of flour (as computed in USDA database, 2008) Mean values for daidzein per gram of soy flour 1500µg/g of flour (as computed in USDA database, 2008) Only the soya portion was responsible for genistein and daidzein compounds in the flour 24/08/2016 9

10 Cell Culture work - Reporter Gene Assays Used previously developed cell lines Cells sub cultured in DMEM media, analyses done in hormone deplete media Cells seeded in 96-well plates Incubated 37 o C 5% CO 2 for 24hrs Expose cells to Phytoestrogens for 24hrs (MMV-Luc cell line) or 48hrs (TARM-Luc cell line) at 37 o C 5% CO 2 Supernatant discarded, cells washed in PBS prior to lysing and bioluminescence read 24/08/

11 Cell line Original Change compared to original Receptors Main mode Source Reference cell line cell line of action MMV-Luc MCF 7 Transfected the MCF-7 ER α an ER β Transcription Dr Marc Muller Willemsen et al.,2004 cell line (Sub- type: with the MMTV Luc Plasmid activation of Liege Luminal) University TARM Luc T47D T47D had psv-ar 0 AR α and AR β Belgium Cell Line (Sub- type: expression vector that encodes Luminal) for an androgen receptor gene Transfected into it, in addition, to the MMTV-Luc plasmid 24/08/

12 Baby food sample (Ig) Reconstitution 20ml acetate buffer 2M, ph 5.2 Enzymatic hydrolysis with(0.3ml Helix pomatia preparation H5 type, sigma units /20ml water), 16h, 52 o C Liquid liquid extraction using 2x10 ml Diethyl ether Organic layer Aqueous layer Acid hydrolyse, 3.2 ml HCl 35%, 2h, 80 C Liquid liquid extraction (2x10 ml Diethyl ether) Organic layer Reverse Phase SPE C18 Wash (5ml water+5ml cyclohexane) Elute (5+2) Methanol wa Normal Phase SPE Wash (10ml ethyl acetate/cyclohexane 30/70 v/v) W 24/08/2016 Elute (10ml ethyl acetate /cyclohexane/ethanol 80/10/10 v/v/v) 12

13 Figure2 Flow diagram of PE Extraction Procedure (adapted from Antignac et al., 2009) 24/08/

14 Aims Exposure assessment (Hospital based nutritional survey) measure flour intake for estimation of exposure to Phytoestrogens; genistein and daidzein The goal of having exposure study alongside Reporter Gene Assays is to be able to calculate cumulative exposure to estrogenicity: 24/08/2016 9

15 Exposure assessment 1. Cumulative exposure to estrogenicity,to the babies, due to genistein and Daidzein assuming additivity of effect is estimated by summing the relative estrogenicity for individual estrogenic dietary phytoestrogens using the formula: EQ = (EC i x EP i ). Where EC i is exposure to chemical i and EP i is its estrogenic potency relative to a standard (17 β- estradiol) (Thomson,2009) 24/08/

16 Estrogenic Equivalence using HBMOS Equation (estrogenic equivalence): XEQ = (intake x REP x 10-6 x A: T x body weight)/serum in litres Where; REP=relative estrogenic potency A: T= measured serum level/theoretical serum level, based on food intake /serum volume where not known can use data in literature (Thomson, 2009) Body weight taken as upper mean of the tested population (9.92) Upper mean serum levels 595ml Adapted from (Thomson, 2009) 24/08/

17 Results 24/08/

18 Figure 1-2 The dose response standard curves of 17β-estradiol. The MMV cell line was treated with increasing concentration of the hormone and luciferase activity measured after 24h.Values are % maximal luciferase activity ± SEM (normalised to 100%). Means of at least three independent experiments (n =3) with each experimental point performed in triplicate. 24/08/

19 Figure 1-3 The dose response standard curves of b) genistein c) daidzein. The MMV-Luc cell line was treated with increasing concentration of the compound and luciferase activity measured after 24h.Values are % maximal luciferase activity ± SEM (normalised to 100%). Means of at least three independent experiments (n =3) with each experimental point performed in triplicate. 24/08/

20 The % relative potency for each of the PEs was calculated as: (EC 50 for the hormone/ EC 50 for the PE) x 100 The dose response curves were plotted by means of triplicate readings for a minimum of three experiments. The EC 50s (M) of estradiol, genistein and daidzein were 4.5 x 10-11, 2.1 x 10-6 and 3.3 x 10-5 respectively. The relative estrogenic potencies for the two compounds were and Consequently the % relative potencies of daidzein and genistein compared to that of 17β-estradiol which is assigned 100 were and /08/

21 The androgenic activity in daidzein equivalents of the different flour blends as determined by combining the extracts with hormone testosterone. Blend SP 1 SP 2 SP 4 SP 3 SP 5 DEQ ± ± ± ± ± 8.4 Blend SP 6 SP 7 SP 8 SP 9 SP 10 DEQ 314 ± ± ± ± ± Table 6 24/08/

22 Bioactivity in daidzein equivalents (DEQ) Mean SEM Type of flour blend extracts. Control SP 1 SP2 SP 4 SP 3 SP 5 SP 6 SP 7 SP 8 SP 10 Figure 17 24/08/

23 Flour Mass FD Mean ±SD FFQ Mean ±SD (Mean FFQ/M ean FD) Ratio Pearson correlat ion r Level of quartile agreement % sam e ± 1 OPP 2 ĸ- statis tic Flour mass 19.21± ± ** Table 7 24/08/

24 Two of the flour samples had estrogenic effect below detectable levels. 4 had barely detectable level 3 had partial agonist effect. The pure soya had the highest effect (0.017) but its effect was disregarded given that it is never consumed on its own. 24/08/

25 The mass of genistein and daidzein in ng/kgbw are converted to 17 β-estradiol equivalents by direct multiplication of upper mean exposure levels by the relative potency. This was for all the population of gruel takers and soya consumers in particular. The sum of the upper means in ng/kgbw of 17- estradiol equivalents for genistein and daidzein translates to and ng/kgbw respectively. This is less than the ADI of 50ng/kgbw 24/08/

26 Genistein (For all gruel consumers) Daidzein (For all gruel consumers) Total Genistein (For soya consumers) Daidzein (For soya Consumers) Total Relative estrogenic potency relative to 17 β-estradiol (EPi) x 10 6 Mean Exposure level ECi taken from upper mean exposure in µg/kgbw/day (adjusted for FFQ overestimation ) A:T ratio) (966.40) (828) XEQ(µg/L) (adjusted for FFQ overestimation ) (2.11 x 10-3 ) (1.69 x 10-3) (3.78 x10-3) (3.02 x 10-3 ) (5.73 x 10-3 )(4.59 x 10-3 ) (1727) (3.62 x 10-3) (2.9 x 10-3 ) (1480) (6.48 x 10-3 ) (5.18 x 10-3 ) (1.03 x 10-2 ) (8.21 x 10-3 ) Table 11 24/08/

27 Given that the EC β-estradiol is 4.5 x M which is equal to 0.012ng/ml a total estrogenic effect due to daidzein and genistein for the soya based gruel consumers of (0.0053ng/ml) would give a less than half agonist effect. These values are above the detectable estrogenicity of ng/ml and hence can be considered to be of partial agonist proportions. 24/08/

28 Conclusions continued Amounts of DEN and GEN consumed by babies was > 1mg/kgbw TDI The two PEs combined had a total of and ng/kgbw for all gruel consumers and the soya based porridge consumers respectively The HBMOS revealed mean estrogenicity of partial agonist proportions 24/08/

29 Conclusions continued CAUTION to parents to use soya flour in moderation especially boy infants could be at higher risk. The study, however, takes cognisance of the fact that the observations were made based on in vitro assays and hence further research is recommended. Recommendations for further studies Animal models- Feeding studies Other in vitro tests 24/08/

30 Acknowledgement Commonwealth Scholarship Commission UK Queens University Belfast Egerton University, Kenya 24/08/

31 Thank you All for listening 24/08/