Metals and epigenetics COPHES March 12, 2012

Karin Broberg Palmgren

- Occupational and Environmental Medicine, Lund University - Institute of Environmental Medicine Karolinska Institutet Why Genetics and Metals?

. Clear susceptibility to toxic effects of several toxic metals. Genetic mechanisms ?

. Beryllium . Lead (Pawlas 2011; Rentschler 2011; Zheng 2011) . Mercury (Custodio 2004, 2005; Engström 2008, 2012; Harari 2011) . Cadmium (Rentschler, manuscript) . Arsenic (Engström 2007, 2008, 2011; Gardner 2012, Schlebusch subm.) . Manganese (Rentschler 2012)

. Ideal for studies of gene-environment interaction - good biomarkers of exposure - well-characterized and fairly simple metabolism - probably the metabolism is a susceptibility factor for several of the metals -> i.e., a slower metabolism, more toxic effects

23 March, 2012 2 Arsenic

Bangladesh

Argentina, Andes Mountains

Occurrence of arsenic in drinking water WHO standard <10 microg/L >100 million people affected

3 Arsenic metabolism

Water: Inorganic arsenic MMA=monomethylated arsenic

DMA=dimethylated arsenic Metabolism

More DMA – faster excretion Inorg.As MMA DMA in urine

MMA trivalent – considered 1-10 µg/l most toxic metabolite in urine Metabolism differs for arsenic

- The fractions inorganic arsenic /MMA /DMA differs between individuals/populations/species

Genetic factors? From Vahter

Argentina Bangladesh 5 E.g. DNA-methylation

6 AS3MT arsenic +3 oxidation state methyltransferase

E.g. DNA-methylation

AS3MT

7 *

AS3MT gene – arrows depict single nucleotide polymorphisms, e.g. at * a C/T exchange

(individual combinations: CC, CT, TT)

8 AS3MT genotype and MMA in urine

Argentina: N=172 women Bangladesh: N=384 pregnant women Average arsenic 200 microg/L Average arsenic 100 microg/L

C-variant=71% C-variant=38%

Low MMA and high DMA= fast and less toxic metabolism

AA AC CC AA AC CC

AS3MT rs3740400 AS3MT rs3740400

Six AS3MT polymorphisms gave a similar pattern in both populations (p<0.05 for all) Engström et al. 2007, 2011 EHP Gene expression of AS3MT in blood for different genotypes

Engström et al. 2011 10 Fast metabolising genotype Much more common In Andes Mountains than in Bangladesh

Argentina (San Antonio de los Cobres) – exposure for thousands of years Bangladesh – appr. 30 years

Hypothesis: There is genetic selection for arsenic- resistant genotypes in areas with arsenic exposure for many generations

11 San Antonio de los Close AS3MT Cobres to Salta Colombia, Haplotype (Arg) (Arg) Mexico Peru

2N= 646 46 50 194 Fast metabolism CTA (%) 68.7 36.7 14.3 50.5

Slow metabolism GCG (%) 25.8 36.4 66.8 39.7

Individuals in San Antonio differ in AS3MT haplotype frequency compared to other native populations (p=10-5 to 10-16) with a presumed lower historically arsenic exposure

Do they differ in general?

Schlebusch et al., to be published 12 Based on 671 microsatellite markers all over the genome The population in SAC differs genetically for arsenic metabolism, but not in general, compared to other native populations in South America

Indicating selection for arsenic tolerance genotype

Schlebusch et al., submitted 13 Why epigenetics and metals?

. How can the environmental changes result in quick and permanent response in an organism?

. How can exposures during early development cause disease much later in life?

. How can carcinogenic compounds be carcinogenic without being mutagenic?

….questions raised for metals ACTGGGTCACTGACG GGGTCCCAAACGT ….. About 3 billion basepairs/ simple DNA set-up

Same genetic code in all cells

Epigenetics

Mechanisms that control the gene expression without changing the gene sequence - in a potentially heritable way

.

Epigenetik är som formatering i ett textbehandlingsprogram

Epigenetik är som formatering i ett textbehandlingsprogram

är som Epigenetik formatering i ett textbehandlingsprogram CGCGATGCGATTTAA Methylated Cytosines before a Guanine

CH3 CGCGATGCGATTTAA

Epigenetic mechanisms 1. DNA-methylation Methylated Cytosines before a Guanine

Expressed gene Not expressed

CH3

CGCGATGCGATTTAA CGCGATGCGATTTAA

From Esteller, Nat Biotech, 2010 How is the DNA-methylation pattern inherited from cell to cell?

DNA-methyltransferases DNA Methyltransferases Methylates hemimethylated DNA DNMT1 (maintenance) DNMT3A and DNMT3B (de novo) Aberrant methylation – clearly linked to cancer Different tumors and subtypes have different DNA-methylation profiles Epigenetic processes essential for orchestrating the development Epigenetic processes essential for orchestrating the development

Toxic compound Inheritance and the environment:

Changes in the environment can change the gene expression through epigenetic regulation Factors in the diet influence DNA-methylation

DNA methylation (DNMT1, DNMT3B)

Folat Kolin Protein Vitamin B12

Arsenic Cadmium

Arsenik very toxic and carcinogenic – but how?

. Arsenic ?? Cancer (skin, lung, bladder) Cardiovascular disease Reduced immune defense Child development Etc.

. Arsenic Gene expression Cancer etc. Epigenetic mechanisms?

29

Arsenic and cadmium via food and drinking water – epigenetic effects in humans?

Bakhtiar Hossain (LU) Karin Engström (LU) Karin Broberg Palmgren (LU/KI) Marie Vahter (KI)

30 High arsenic in drinking water in some parts of the Andes Mountains

San Antonio de Los Cobres about 200 ug/L

31 San Antonio de Los Cobres 3800 m a s 5000 inhabitants Interviews Water, blood and urine samples Targets DNA-methylation

• Global methylation of DNA repeats of “jumping genes” LINE1 (should be methylated and not expressed) (N=202) LINE1 hypomethylation associated with bladder cancer risk • Gene-specific methylation – tumor suppressor gene p16 (should be unmethylated and expressed) (N=202) p16 mutation or methylation found in tumors • Methylation on specific sites – N=450,000 (N=90)

• Further: Arsenic in blood and urine • Gene expression –N~ 25,000 transcripts (N=90)

Arsenic in blood/urine is associated with more p16 methylation and with less expression in blood.

Urine Beta =0.14 (95% CI 0.050-0.24) p=0.0028 Blood Beta=0.19 (95% CI 0.077-0.31) p=0.0012

Arsenic in urine (rs=-0.20 p=0.066) and blood (rs=-0.27 p=0.020) correlate with p16 expression

No association between arsenic and LINE1

Hossain et al, submitted AS3MT determines arsenic metabolism efficiency – does it influence toxic effects?

Effect of arsenic in urine on p16 methylation stratified for AS3MT genotype

AS3MT genotype p16 methylation

Mean (SD) β (95% CI) p CC (N=93) 2.57 (0.92) 0.13 (-0.0038 to 0.27) 0.023 AC (N=61) 2.89 (0.93) 0.19 (0.040 to 0.34) AA (N=15) 3.29 (0.89) 0.32 (-0.52 to 1.16)

Slow metabolisers – more p16 methylation Low p16 methylation – protected from arsenic?? AS3MT chromosome band 10q24, depending on AS3MT genotype - differently methylated - results from the 450K chip (Engström, manuscript in prep.)

AS3MT .genotype (A/C) modifies methylation of AS3MT .modifies gene expression (C less expressed) .modifies arsenic metabolites i urin .modifies toxic effects – epigenetic alterations of p16

36 Zn C Zn d

. Cadmium mimics zinc

. Carcinogen (lung, endometrium, breast, prostate?) but not a

strong mutagen . Effekts on methylation in vitro . Inhibits DNA methyltransferases in vitro

. In vivo? N=202 kvinnor LINE1 methylation in blood Cd i urine= median 0.23 ug/L Cadmium (from food) – similar levels as for Swedish women

LINE1 (500,000/genome) – a proxy for global methylation – should be hypermethylated (blood, healthy population) A1 : TTT/CGTGGTGT/CGTT/CGTTTTTTAAGTT/CGGTTT 87% 87% 86% 86% 500 400 300 200 100 0 E S A T C A G T G T G T C A G T C A G C T T A G T C T G 5 10 15 20 25

LINE1 (A549, lung cancer cell line)

D1 : TTT/CGTGGTGT/CGTT/CGTTTTTTAAGTT/CGGTTT 47% 70% 63% 72%

400

300

200

100

0

-100 E S A T C A G T G T G T C A G T C A G C T T A G T C T G 5 10 15 20 25 Low-level environmental cadmium exposure is associated with DNA hypomethylation

p=0.0070, adjusted p=0.026

Cadmium is negatively associated with DNA-methyltransferase gene expression in blood. Hossain et al, EHP 2012

DNA-methyltransferases modifies the epigenetic effect of cadmium on LINE1

Genotype β (95%CI) p DNMT1 rs10854076 CC (N=25) -1.09 (-2.02 to -0.15) 0.041 CG (N=69) -0.96 (-1.68 to -0.24) GG (N=78) -0.052 (-0.58 to 0.47)

DNMT1 rs2228611 GG (N=35) -1.19 (-1.99 to -0.40) 0.050 GA (N=68) -0.38 (-1.07 to 0.31) AA (N=69) -0.10 (-0.67 to 0.47)

Individuals with a rarer genotype have less methylation with increasing cadmium exposure

EHP Jan 2011 N=512 elderly men

Hypomethylation of LINE1 with patella lead. 41 Summary…

. The metabolism for arsenic differs between individuals depending on AS3MT genotype . The AS3MT fast-metabolising genotype is more common in an area with a long historical arsenic exposure – selection?

. Arsenic and cadmium is associated with premalignant epigenetic effects - for arsenic the epigenetic effects modified by the AS3MT genotype - for cadmium the epigenetic effects modified by the genetic background for the DNA methylating machinery

- Lead and nickel epigenetic effects – other metals? Thanks to - Sweden: Prof. Marie Vahter KI, Dr. Gabriela Concha, National Food Administration Dr. Karin Engström, Dr. Bakhtiar Hossain, Occupational and Environmental Med., Lund University Dr. Mattias Jakobsson, Dr. Carina Schlebush, Evolutionary Biology Centre, Uppsala University Bangladesh: Assoc. Prof. Rubhana Raqib, ICDDR,B,

Thank you!