Modulation of a LMB CYP3A Isoform’s Expression By

Endocrine DisruptorsRicardo Lugo

PI: Dr. David Barber

30 July 2004 Lugo - Cytochrome p450 2

Overview

• Pesticides: the ongoing problem• Fish as a Model Organism• P450’s Role in Metabolism• Endocrine Disruption• Hypothesis• Brief Methods• Results

30 July 2004 Lugo - Cytochrome p450 3

Lake Apopka

30 July 2004 Lugo - Cytochrome p450 4

Steroid Hormones – Control Site

Spring

Summer

Estradiol

10

100

1000

10000

10 100 1000 10000

10

100

1000

10000

10 100 1000 10000

Estradiol

Testosterone

Testosterone

30 July 2004 Lugo - Cytochrome p450 5

Steroid Hormones – Impacted Site

Spring

Summer

10

100

1000

10000

10 100 1000 10000Testosterone

Estradiol

10

100

1000

10000

10 100 1000 10000

Testosterone

Estradiol

30 July 2004 Lugo - Cytochrome p450 6

Pesticides’ Effect on Fish

• Reproductive difficulties• Transcription of CYP3A68

varies in presence of DDE– 12x in Female– 12x in Male

• Differences by sex may imply hormonal modulation of transcription

• CYP3A69 expression not altered– Perhaps due to different

promoter region from CYP3A68

20

15

10

5

0

-5

-10

-15

-20 3A68 3A69

FemaleMale

Fold

Ch

an

ge in

Ste

ad

y S

tate

m

RN

A

LMB CYP3A Expression

30 July 2004 Lugo - Cytochrome p450 7

Fish as a model

• Swim in water where contaminants are found• Sensitive to environmental contaminants• Populations may be impacted• Top of food chain – predatory fish• Game fish which are eaten by people• Complex system– good model for human health

(Large Mouth Bass)

30 July 2004 Lugo - Cytochrome p450 8

Humane Treatment of Fish

• Euthanize fish to obtain Genomic DNA from Liver• File procedure with IACUC

– Problem with standard procedure is that anesthesia (MS 222) may interfere with our studies

– Obtained permission to knock them out without anesthesia

• Take the LATA Training module: “The Humane Care & Use of Laboratory Fish”

30 July 2004 Lugo - Cytochrome p450 9

Cytochrome P450• Problem

– Lipophilic, toxic xenobiotics accumulate

• CYT P450’s role– convert to more

hydrophilic metabolites, detoxify

– Phase I Biotransformations• Reduction, Oxidation,

Hydrolysis• Prepare for Phase II• Detoxify active compounds

• CYP3A involved in metabolism of sex steroids http://quanta.synchem.kyoto-u.ac.jp/bioquant/bio.html

30 July 2004 Lugo - Cytochrome p450 10

Endocrine Disruption• Alters the function of the

endocrine system – With adverse effects on

health of organism and progeny

• Potential Mechanisms– Direct: disrupt hormone

receptor– Indirect: modulate

concentration of hormone

• Too much / too little 3A enzyme– Hormone metabolized at

faster / slower rate– Results in less / more

hormone in organism

Direct

Indirect

http://www.emcom.ca/EM/humans.shtml

30 July 2004 Lugo - Cytochrome p450 11

Hypothesis• Pesticides act as indirect hormone disruptors, cause

adverse disruption of normal physiologic function– Modulate levels of Cytochrome P450 3A isoform– By regulating transcription of CYP3A gene

• Sequence promoter regions of CYP3A68 and CYP3A69– 3A68 promoter will contain ERE or similar Response

Element (RE)– 3A69 promoter will not

30 July 2004 Lugo - Cytochrome p450 12

Isolating the Promoter

Sequence from plasmidUsing M13 FP / RP primers

Isolate PlasmidPromega Wizard SV Plasmid Miniprep

Clone isolated promoter DNAPromega pGEM-T Easy Cloning

Sequence from PCRRequires specialized primers

Amplify DNA upstream of TSS for CYP3A68 & 69Genome Walker PCR

Obtain, Purify, Digest Genomic DNA

Isolate Liver

Large Mouth Bass

30 July 2004 Lugo - Cytochrome p450 13

Genome Walking Prep• Isolate Genomic DNA

from Bass Liver• Digest with four

different restriction enzymes

• Ligate the GenomeWalker Adapter to digested DNA

Undigested

Genom

ic DN

A

Digested Genomic DNA

10kb6kb4kb3kb2kb

1kb

10kb6kb4kb3kb2kb

1kb

® 2000 Genome Walker Protocol

30 July 2004 Lugo - Cytochrome p450 14

Genome Walking PCR• Primers designed to

transcribe upstream• 1º PCR:

– Amplify larger product

• 2º PCR:– Amplify smaller product,

closer to promoter– Cut band for cloning

• CYP3A68 – ~1.2kb DNA strand

• CYP3A69 – ~3kb DNA strand

10kb6kb4kb3kb2kb

1kb

® 2000 Genom

e Walker

Protocol

30 July 2004 Lugo - Cytochrome p450 15

Cloning the DNA1) Ligate insert into pGEM-T

Easy vector2) Transform JM109 E. Coli

cells3) Plate cells, Blue/White

screening4) Grow white colony culture5) Plasmid miniprep6) Check with M13FP/RP PCR

- Initially, problems growing colonies

- With new primers, CYP3A68 cloned

CYP3A68 did clone

CYP3A69 did not

10kb6kb4kb3kb2kb

1kb

30 July 2004 Lugo - Cytochrome p450 16

Problem: AP2 Everywhere

• Sequencing showed Genome Walker Adapter bound to both ends of 3A68 DNA

• GSP2 / AP2 strands should be favored amplification

• AP2 / AP2 strands amplifying– Perhaps libraries too old,

contaminated

• PCR with and without GSP2 to determine valid bands– 3A69 amplifying properly,

send for sequence– Redigest genomic DNA with

different digestion enzymes

3A

68

w

/GS

P2

+A

P2

3A

68

w/A

P2

on

ly

3A

69

w

/GS

P2

+A

P2

3A

68

w/A

P2

on

ly

10kb6kb4kb3kb2kb

1kb

30 July 2004 Lugo - Cytochrome p450 17

** TFSEARCH ver.1.3 ** (c)1995 Yutaka Akiyama (Kyoto Univ.)Database: TRANSFAC MATRIX TABLE, Rel.3.3 06-01-1998 Query: 3a-b ap2 sequence (775 bases)TFMATRIX entries with High-scoring:

1 CCCGGGCTGG TCCTTTGCAG GTGTGTTCTA ATTTTTTGAG ATACTGATTT entry score ----------> M00076 GATA-2 89.3 ---------> M00077 GATA-3 88.1

51 TGGGGTTTTC ATTAGTTGTC AGTTATAATC ATCAAAATTA AAAGGAATGA entry score ------> M00101 CdxA 88.6 -------------> M00137 Oct-1 88.0

101 ACACTTGAAA TATATCAGTC TGTGTGGAAT GAATGTATAC ATTATACAAG entry score <------ M00240 Nkx-2. 100.0 --- M00258 ISRE 88.7

151 TTTCACTTTC TGAATGGAAT TACTGAAATA AATAAACTTT TTGCTGATAT entry score -------> M00101 CdxA 93.6 ------------> M00258 ISRE 88.7 ---- M00099 S8 88.2

201 TCTAATTATA TGACCAGCAC CTGTAAATAA AACAAATAAA TAAAAAAGTT entry score -------> M00148 SRY 100.0 ------> M00101 CdxA 98.6 ---------------> M00267 XFD-1 90.0 ------------> M00099 S8 88.2

251 AAAAAGCAAT TTCCATGAAA ACACAATTGG TTAACTCTGA GCTCAGAACA entry score -------> M00148 SRY 92.7

301 TTTCCATTCA AACTGTGATT ACCACAGTAA TGCACAGTTC AAATTACATC entry score

351 TTTTCATGAA CATGGTGAAC ATGACCCAAT TTCAGTAGAA AAGATTGTAG entry score

401 CCATTATAAA GGCCCCACGA GGCTTTAATA ATCAGATTAC AACCACAATA entry score ------> M00101 CdxA 92.9 -------------> M00137 Oct-1 92.0

451 AACTTGGAAA GAAGTTTGGA ATAAGGTAAA TCAAAAGAGG TTATTCTCTA entry score -------> M00148 SRY 90.9

501 ACATGCTAAT ATTAGCGTGC ATAACTAGCC AACAGTCCAG CAGGGTGACT entry score ------- M00173 AP-1 88.7

551 ACGCCACACA GGGAGCCACT GCTAGTTCAG CTAACACATA CATTTAAAAG entry score ---> M00173 AP-1 88.7 ------> M00100 CdxA 88.5

601 GACTGGGATT ATTCTATATT TTCCTTATGG TCAACAAATC CAATGGAAAG entry score

651 ACCAAAACCA AATTTTTTTT AATTCTACCT AACCTGTCTG TGGCATTCAG entry score ------> M00101 CdxA 92.9 ---- M00053 c-Rel 90.1

701 TACCATAGCA AATGTTACTC AAATAGGAAT AAATAGCTGC AATAAATACA entry score -- M00241 Nkx-2. 88.2

751 TAATTTGGTG TTTGGTGCTC TAGTG entry score -----> M00241 Nkx-2. 88.2

3A69 Promoter Analysis

Notable TFs:- GATA-2/3- CdxA- Nkx-2- ISRE

- SRY

- XFD-1- AP-1- c-Rel

http://www.cbrc.jp/research/db/TFSEARCH.html

30 July 2004 Lugo - Cytochrome p450 18

Acknowledgements

• Dr. Barber• Dr. Wood• Ms. Copeland• Ms. Douglas-

Nikittan

• Alex McNally• Tamece Knowles• Kathy Childress