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Modulation of a LMB CYP3A Isoform s Expression By Endocrine Disruptors Ricardo Lugo PI: Dr. David Barber.

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Presentation on theme: "Modulation of a LMB CYP3A Isoform s Expression By Endocrine Disruptors Ricardo Lugo PI: Dr. David Barber."— Presentation transcript:

1 Modulation of a LMB CYP3A Isoform s Expression By Endocrine Disruptors Ricardo Lugo PI: Dr. David Barber

2 30 July 2004Lugo - Cytochrome p4501 Overview Pesticides: the ongoing problem Fish as a Model Organism P450s Role in Metabolism Endocrine Disruption Hypothesis Brief Methods Results

3 30 July 2004Lugo - Cytochrome p4502 Lake Apopka

4 30 July 2004Lugo - Cytochrome p4503 Steroid Hormones – Control Site Spring Summer EstradiolEstradiol EstradiolEstradiol Testosterone

5 30 July 2004Lugo - Cytochrome p4504 Steroid Hormones – Impacted Site Spring Summer Testosterone EstradiolEstradiol EstradiolEstradiol

6 30 July 2004Lugo - Cytochrome p4505 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 Female Male Fold Change in Steady State mRNA LMB CYP3A Expression

7 30 July 2004Lugo - Cytochrome p4506 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)

8 30 July 2004Lugo - Cytochrome p4507 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

9 30 July 2004Lugo - Cytochrome p4508 Cytochrome P450 Problem –Lipophilic, toxic xenobiotics accumulate CYT P450s 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

10 30 July 2004Lugo - Cytochrome p4509 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

11 30 July 2004Lugo - Cytochrome p45010 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

12 30 July 2004Lugo - Cytochrome p45011 Isolating the Promoter

13 30 July 2004Lugo - Cytochrome p45012 Genome Walking Prep Isolate Genomic DNA from Bass Liver Digest with four different restriction enzymes Ligate the GenomeWalker Adapter to digested DNA UndigestedGenomic DNA Digested Genomic DNA 10kb 6kb 4kb 3kb 2kb 1kb 10kb 6kb 4kb 3kb 2kb 1kb ® 2000 Genome Walker Protocol

14 30 July 2004Lugo - Cytochrome p45013 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 10kb 6kb 4kb 3kb 2kb 1kb ® 2000 Genome Walker Protocol

15 30 July 2004Lugo - Cytochrome p45014 Cloning the DNA 1) Ligate insert into pGEM-T Easy vector 2) Transform JM109 E. Coli cells 3) Plate cells, Blue/White screening 4) Grow white colony culture 5) Plasmid miniprep 6) Check with M13FP/RP PCR -Initially, problems growing colonies -With new primers, CYP3A68 cloned CYP3A68 did clone CYP3A69 did not 10kb 6kb 4kb 3kb 2kb 1kb

16 30 July 2004Lugo - Cytochrome p45015 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 3A68 w/GSP2+AP2 3A68 w/AP2 only 3A69 w/GSP2+AP2 3A68 w/AP2 only 10kb 6kb 4kb 3kb 2kb 1kb

17 30 July 2004Lugo - Cytochrome p45016 ** 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 l

18 30 July 2004Lugo - Cytochrome p45017 Acknowledgements Dr. Barber Dr. Wood Ms. Copeland Ms. Douglas- Nikittan Alex McNally Tamece Knowles Kathy Childress

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