low doses of hg and of estrogenic disruptors in flanders ... · total hg and having received breast...

Low doses of Hg and of estrogenic disruptors in Flanders:

consequences for health

Prof. Dr. Willy Baeyens

Analytical and Environmental

Chemistry (ANCH) 1Herhaling titel van presentatie

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Ancient Hg cycle

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Analytical and Environmental Chemistry (ANCH)


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Modern Hg cycle

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HEALTH EFFECTS

� Neurotoxic (NRC, 2000; Rice et al., 2003; Debes et al., 2006; Counter and Buchanan, 2004; van Wijgaarden et al., 2006)

� Possibly carcinogenic (IARC, group 2B)

� Damage to lungs and kidneys (Cardenas et al., 2003; Counter and Buchanan, 2004 )

� Cardiovascular diseases (Stern, 2005)

� Eye irritation and allergic reactions (Hazardous Substances Data Bank)

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FLEHS II

Exposure to mercury in the general Flemish population: Results of the 2nd Flemish Environment and Health Study

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www.milieu-en-gezondheid.be

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Human biomonitoring

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Contaminants

in the

Environment

Possible

adverse

health effects

Human

Biomonitoring

Exposure ResponseDose

Biomarker of

exposure

Biomarker of

effect


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Study design

� 3 age groups:– 250 newborns and mothers – maternities

– 200 adolescents (14-15 jaar) – schools

– 200 adults (20-40 jaar) – provincial institutes

� Multistage sampling strategy

� Selection criteria: – Living 10 years in Flanders

– Being able to fill in a questionnaire

– Written informed consent

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Output

� Reference values – Geometric means (95% CI) and 90e percentiles

– Adjusted for a priori defined confounders

� Descriptive statistics per age group– Personal factors (sex, age,…)

– Lifestyle (nutrition, smoking,…)

– Environmental (urban, traffic,…)

� Time trends: comparison earlier values (2002-06)

� Comparison guidelines and international literature

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Response rate

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Newborns Students Adults

N 255 210 204

% response - 51,8% 30,0%

% participation 88% 69,5% 92,3%

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Study population

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Mothers of

newborns

Students Adults

N 255 210 204

Age(yr)Geometric mean(min.-max)

30,3 (18,2 – 42,4) 14,8 (13,8 – 16,3) 33,9 (21,4 – 40,6)

% male / female 0 / 100 57,6 / 42,4 47,1 / 52,9

% smokers 11,6 13,6 23,3

% higher education 61,5 56,0 (family) 77,5

% urban 61,6 36,7 48,5

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Region Participants THg (µg g-1) Reference

Flanders adolescents 0.19 (P10-P90

0.07-0.47)

Our Data

Flanders mothers 0.34 (P10-P90

0.12-

0.86)

Our data

Slovenia mothers 0.38 (P10-P90

0.07-0.78)

Miklavcic et al., 2011

Sweden mothers 0.22 (Min-

Max 0.04-

0.83)

Gerhardsson and Lundh,

2010

Northern part of the Persian Gulf fishermen 2.9±2.2 Agah et al., 2010

Guizhou Province, China mining area 45±22 y

(m/f)

4.6±2.5 Li et al., 2009

Florida, US mothers 0.25 (95% CI

0.22-0.28)

Karouna-Renier et al.,

2008

North Atlantic, Faroe Island

-Women consuming whale

-Women not consuming whale

26-30 y

3.03

1.88

Weihe et al., 2005

Brazilian Amazon women 41y 10±4.7 Passos et al., 2003

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Adolescents

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0

0,1

0,2

0,3

0,4

low medium high

Hg

-T a

nd

MM

Hg

(µ

g g

-1)

Fish consumption level

Adolescents: Hg in hair versus fish consumption

Hg-T

MMHg

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Mothers

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0

0,1

0,2

0,3

0,4

0,5

Hg

-T a

nd

MM

Hg

(µ

g g

-1)

low medium high

Fish consumption level

Mothers:Hg in hair versus fish consumption

Hg-T

MMHg

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Methylmercury

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PTWI = Provisional Total Weekly Intake

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Dose effect relations in adolescents

Significant negative associations between:

� total Hg and MMHg levels and asthma ever reported (p = 0.0481 for Hg-T and p = 0.0455 for MMHg)

� total Hg and having received breast feeding as a newborn (p = 0.0061) and to age at menarche in girls (p = 0.0124)

� MMHg and allergy for animals (p = 0.0079) and between the logarithm of MMHg and free testosterone levels (p=0.019)

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Ranking of significant gen-mercury exposure correlations-Boys

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Exposure gen CC n p-value

HG_T JAK2 .2570 N=102 p=.009

HG_MM JAK2 .2640 N=96 p=.009

HG_MM ARID4A .2413 N=100 p=.016

HG_T ARID4A .2291 N=106 p=.018

HG_T Hist1H4L -.2013 N=107 p=.038

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Ranking of significant gen-mercury exposure correlations-Girls

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Exposure gen CC n p-value

HG_T HLAdrb5 -.2988 N=79 p=.007

HG_T PIAS2 -.2628 N=83 p=.016

HG_MM HLAdrb5 -.2681 N=78 p=.018

HG_T MAN1B1 -.2628 N=79 p=.019

HG_MM PIAS2 -.2466 N=82 p=.026

HG_MM MAN1B1 -.2502 N=78 p=.027

HG_MM GIT -.2444 N=79 p=.030

HG_T ABCA1 -.2220 N=79 p=.049

HG_MM ABCA1 -.2233 N=78 p=.049

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Role of some genes

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� The JAK2/STAT3 axis is a major transducer of HN-mediated neuroprotective activity.

� HLAdrb5 plays a central role in the immune system by presenting peptides derived from extracellular proteins. It has also been reported to be involved in genetics of Parkinson Disease (Sassi, 2011).

� GIT1 was examined in 192 children with ADHD and 196 children without the condition, and it was found that a specific variation in the gene was more than twice as common among the affected children

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Introduction EDCs

� Endocrine Disrupting Compounds

� Possible effects: change in development, lower fertility, diabetes, obesity, cancer, …

� Non-linear dose-response curve (low dose effect)

� Synergetic or antagonistic effects

� Man made or natural

� Broad range

� Many unknown

Molecule specific techniques insufficient

Receptor based technique � overall endocrine disrupting activity

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Prof. Dr. Willy Baeyens, M. Sc. Tara Vandermarken & Dr. Sandra De Galan

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ERE-CALUX bioassay

20

Estrogen Responsive Elements - Chemically Activated LUciferase gene eXpression

18/04/2013Prof. Dr. Willy Baeyens, M. Sc. Tara Vandermarken & Dr. Sandra De Galan


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ERE-CALUX bioassay

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� Relative method � use of 17β-estradiol (E2)

� Full dose-response curve

� Fitted by the weighted least square regression

� Described by 4 parameter Hill function

xi: E2 concentration or sample concentrationyi: the response normalized to the maximum response

y0: intercept or experimental background of luminescencem: plateau of maximum inductionh: Hill coefficientk: Effective Concentration on 50% of the maximum induction (= EC50)

εi: remainder term of the i-th observation

BEQ = Biological EQuivalent concentration in pg E2/g sample



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Full dose-response curve

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Materials and Methods

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Sampling:

- Converted vacuum cleaner

- Only use metal and Teflon

- Pre-cleaned glass fiber thimble to collect dust

- 12 kindergarten classes



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Extraction:

- Accelerated Solvent Extraction (ASE)

- Acetone/Hexane 1:1 (v/v)



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Clean-up:

- Florisil column

- Magnesium Silicate

- Vacuum

- EPA method for phthalate esters

- Conditioning: hexane

- Eluation: acetone/hexane 1:9 (v/v)



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Results: Activity

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� Estrogenic activity in % of the 12 different dust samples where 100% is given to the sample showing the highest activity

� Interior score relates to the amount of plastic materials used in the class room

- Type of flooring

- Type of furniture

- Amount of plastic toys

- Presence of electric equipment (computers)

- Presence of plastic play grounds

- Recently cleaned or not



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Relation to interior score

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Relation to phthalate content

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Conclusions

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� Large differences in estrogen activity of dust samples

� Relation between the amount of plastic in the environment (interior score) and measured activity

� Possible prevention

- Frequently cleaning

- Using a HEPA filter on the vacuum cleaner

- Prefer wooden toys or furniture or make sure the plastic used is of good quality (DINP or DIDP as plasticizer)

� Overall activity relates to total phthalate content when subtracting the non-estrogenic compounds like DINP and DIDP

� Using a bioassay = faster, cheaper and will take unknown EDCs, synergetic or antagonistic effects into account



low doses of hg and of estrogenic disruptors in flanders ... · total hg and having received breast...

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