Toxicogenomics – Many many genes…

Way back when… in 2000, microarray was the new wave of technologies to address a scientific challenge that has been around for a long time. If we think  about how an organism responds to an environmental change, disease, or new stage of development there is a corresponding change at the gene expression level. This change in gene expression provides for a host of new proteins that will be needed, and the suppression of all of the proteins that will not be needed.

The challenge was how would we be able to capture hundreds, maybe thousands of these changes… at the same time. We needed a molecular “snap-shot” of all of the mRNAs in a cell before the change, followed by another after the change, and then we needed the ability to sort out the results.

Enter the microarray. The paper referenced above provides a nice overview of the challenges the were faced, and the technologies that were developed to face these challenges.

I have asked the Tox 1401 students to pull out some of the genes mentioned in the paper and take a look at the annotated information about their gene of interest from OMIM and UniProt. If you would like to see their descriptions please move on to the comments section.

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37 thoughts on “Toxicogenomics – Many many genes…

  1. DNA microarrays are a modern step in technology, which has uprooted from the idea to monitor the expression profiles of various cells. The article uses the human breast cancer MFV-7 cells to analogously compare how the technique is applied and used for further research concerning cancer and the pathways it may illicit. In this article specifically, breast cancer cells (MFV-7) are treated with doxorubicin selected for resistance. The results in turn indicated that the treatment of doxorubicin induced changed in the expression of large number of genes in MCF-7 cell. Thus, these genes including microsomal epoxide hydrolase 1, the 26S proteasome regulatory subunit 4, and XRCC1 are involved in repair of DNA strand breaks following exposure to ionizing radiation or alkylating agents that may be functionally relevant for drug resistance. Thus, I looked up epoxide hydrolase 1 gene on the respective databases (OMIM and UniProt) to accurately identify its characteristics.

    Epoxide hydrolase 1- OMIM
    Epoxide hydrolases plays an important role in both the activation and detoxification of exogenous chemicals such as polycyclic aromatic hydrocarbons. Microsomal epoxside hydrolase is a bifunctional protein that plays a central role not only in carcinogen metabolism but is also able to mediate the sodium-dependent uptake of bile acids into hepatocytes. In terms of mapping it is correlated with the loss of human chromosome 6 from each cell line. In a study by Brown and Chalmers (1986) they concluded that the human gene for epoxide hydrolase may be on chromosome 6. Certain observations in hybrid cells suggested that other gene products can affect the level of activity expressed by the cell.

    Epoxide hydrolase 1- UniProt
    The function: Biotransformation enzyme that catalyzes the hydrolysis of arene and aliphatic epoxides to less reactive and more water soluble dihydrodiols by the trans addition of water. It is found in the liver. Its biological process is for Aromatic hydrocarbons catabolism and Detoxification. Defects in EPHX1 are a cause of familial hypercholanemia (FHCA) FHCA is a disorder characterized by elevated serum bile acid concentrations, itching, and fat malabsorption.

    • When reading your post all i could think about was organic chemistry because of the word “epoxide”! Ha! So basically, if a person had a genetic mutation on chromosome 6, this would disrupt the coding for epoxide hydrolase right? As a result of that,exogenous chemicals wouldn’t be detoxified properly, which would lead to deleterious effects in the body. Do you happen to know of any other genes that can carry out this same type of response as epoxide hydrolase or maybe think that if they’re any other genes like this?

  2. I do not fully understand the micro array I’m not going to say I do but what i understand makes it seem like quite a valuable and helpful tool. What allures me the most if the
    actual prep of gene chips or slides. How the actual complimentary DNA is inserted? Once there though they are great. I see this technology helping most with showing up/down regulations of a hopefully extractable, and useful, protien to be “mass produced” to aid the body in replacement of what it might not be able to make more/enough of. The articles mentions something of this sort with xenobiotics. Maybe even a step past that but introduction to allow some of these proteins/genes derived from earlier Dna to become more natural. Sadly under such a guess and check philosophy many pathways can be activated or inhibited so the goal is to find out the most efficient ways to turn certain things on while others off and visa-verse.
    Nrf2 is one of two Nuclear Respiratory Factors. these genes help in the binding and holding together of created DNA. The article mentions combined work done by: R. Hu, C. Chen, V. Hebbar, .A. N. Kong, R. Yu, E. Owuor, R. Ee, and S. Mandlekar on Signal transduction events elicited by cancer prevention compounds and cDNA microarray analysis of sulforaphane and (-)-epigallocatechin-3-gallateinduced gene expression profiles showing that some genes from green tea at low doses can activate the MAPK pathway (regulation of cell communication via receptors in preparation of growth/division) in turn inducing nrf2 yet too much and the cell begins apoptosis to avoid too many of these cell division regulators. In regard to caner these genes are of interest as leveling out these within a person may allow cell division to presume somewhat of a normal pathway.
    Taking a look at OMIM there is mention of how these genes control regulation of energy from the mitochondria into the cell regulating the growth-responsive metabolic pathways of energy transduction, translation, and replication Efiok, B. J. S., Chiorini, J. A., Safer, B propose that alpha-pal is a transcription factor that links the transcriptional modulation of key metabolic genes to cellular growth and development Pubmed mentions its functions as a transcription catalyst using phosphorilization to induce DNA binding the genes are Ubiquitously expressed with strongest expression in skeletal muscle and further adds how the nuclear respiratory factors are Implicated in the control of nuclear genes required for respiration, heme biosynthesis, and mitochondrial DNA transcription and replication

  3. DNA Microarrays contain a large number of genes, but also have a small size. This is very useful to researchers and scientists who want to engage in a large study of genes. Microarrays can be used to examine the expression of thousands of genes all at once. Each spot on a microarray contains multiple identical strands of DNA. As a result, each spot represents one gene. In reference to this scientific paper, I have chosen the gene UFD1L, also known as Ubiquitin fusion-degradation protein. Using both the OMIM and Uniprot databases, I stumbled upon sufficient information pertaining to UFD1L, which is shown below:

    *Uniprot Database:
    When I searched the gene UFD1L on the Uniprot database, there were over 17 different entries that popped up. However, the different entries pertained to different species such as humans, mouse, rats, and fruit flies. I chose the Homo sapiens species (UFD1_HUMAN). The recommended name for this gene species is Ubiquitin fusion degradation protein 1 homolog. The shorter name is UB fusion protein 1. Its protein sequence is a total of 307 AA. This gene is a part of the UFD1 family. It is usually found in adult heart, skeletal muscle, pancreas muscle, and in fetal liver and kidney. UFD1L is an essential component of the Ubiquitin-dependent proteolytic pathway. This pathway is responsible for the degradation of Ubiquitin fusion proteins.

    *OMIM Database:
    Pizutti et al. have done research on DiGeorge syndrome, which involves the deletion of the 22q11.2 regional genes. UFD1L is involved in the degradation of Ubiquitin fusion proteins. Suggested findings have concluded that “the proteolytic pathway recognizing ubiquitin fusion proteins for degradation is conserved in vertebrates and that UFD1L gene hemizygosity may be the cause of some of the CATCH22-associated developmental defects”. Yamagishi et al. have discovered that the human UFD1L gene was deleted in all of the tested 182 patients who had the 22q11.2 deletions.

    http://omim.org/entry/601754

    • What exactly does UFD1L do in the body? What did the study test, like what did they do to the gene to see how it was affected (UV radiation, chemical exposure, etc.)?

    • I too looked up the NRF2 gene. I found it very interesting how you looked into the effects of green tea on the MAPK pathway. My article did not touch upon that, I find it very interesting.

  4. Khew –Voon Chin of the Department of Medicine and Pharmacology and The Cancer Institute of New Jersey, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey in New Brunswick and A.-N. Tony Kong of the Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey in Piscataway wrote a paper on the Application of DNA Microarrays in Pharmacogenomics and Toxicogenomics that assesses gene expression induced by pharmaceutic compounds. One study was on the expression of human breast cancer MCF-7 cells that are treated with doxorubicin or selected for doxorubicin resistance. One of the genes studied in this experiment was X-ray repair cross-complementing protein 1 (XRCC1), a gene that is involved in repair of DNA strand breaks following exposure to ionizing radiation or alkylating agents. Chin and Kong found that XRCC1 may be functionally relevant for drug resistance and that up-regulation or constitutive overexpression of XRCC1 may enhance the repair of breaks of DNA strands and confer drug resistance in tumor cells.
    Through the OMIM database, I found a study that studied the effects of X-Ray radiation on the XRCC1 gene. The study goes over the gene function of XRCC1, and it explains that XRCC1 interacts with human polynucleotide kinase (PNK) in addition to its interactions with DNA polymerase-beta (POLB) and DNA Ligase III (LIG3). Together, these genes repair single-strand breaks typical of those induced by reactive oxygen species and ionizing radiation. This study was conducted in Chinese hamsters, and the study showed that XRCC1 helps repair DNA strands that get damaged by UV radiation by stimulating the DNA kinase and DNA phosphatase activities of PNK at damaged DNA termini, thereby accelerating the overall repair reaction.
    http://omim.org/entry/194360
    The UniProt database also says that XRCC1 corrects defective DNA strand-break repair and sister chromatid exchange following treatment with ionizing radiation and alkylating agents. The database says that this gene is located in the nucleus but accumulates at the site of DNA damage. The UniProt database specifies that the biological processes of XRCC1 are base-excision repair as well as single strand break repair. The specific molecular functions of XRCC1 are damaged DNA binding and protein binding.
    http://www.uniprot.org/uniprot/P18887

    • XRCC1 a dna repair protein,i read that protein encoded by the gene is involved in the efficient repair of DNA single-strand by exposing to ionizing radiation and alkylating agents. one of the questions I have is that what substance is involved in the repair of DNA?how does it repair the broken stand..

  5. The protein encoded by this gene is a basic leucine zipper transcription factor that lacks a transactivation domain. It is known to bind the US-2 DNA element in the promoter of the oxytocin receptor (OTR) gene and heterodimerizes with other leucine zipper-containing proteins. The encoded protein can also form homodimers, and since it lacks a transactivation domain, the homodimer could act as a repressor of transcription. this gene may also be involved in the cellular stress response.
    Uniprot database:
    MRF acts as a transcriptional activator and repressor. It plays role in regulating the lineage-specific hematopoietic by repressing ETS1-mediated transcription of erythroid-specific genes in myeloid cells. Which is required for monocytic, macrophage, podocyte and islet beta cell differentiation. I also read that it activates the insulin and glucagon promoters. MRF binds element G1 on the glucagon promoter and is involved either as an oncogene or as a tumor suppressor, depending on the cell context.
    OMIM database:
    Members of the MAF family appear to play important roles in the regulation of differentiation. By subtractive cloning and differential screening analyses using MCNS patient T lymphocytes, identified a number of genes differentially regulated in MCNS. MAF enables extended expansion of mature monocytes and macrophages in culture without the loss of differentiated phenotype and function. It is possible to amplify functional differentiated cells without malignant transformation or stem cell intermediates.

    • Was the study from the OMIM database on human cells? Can know of any other genes that function like this? If monocytic, macrophage, podocyte and islet beta cell differentiation doesn’t occur properly, what do you think might happen?

  6. XRCC1 a dna repair protein, i read that protein encoded by the gene is involved in the efficient repair of DNA single-strand by exposing to ionizing radiation and alkylating agents. one of the questions I have is that what substance is involved in the repair of DNA? how does it repair the broken stand.

    • The way XRCC! goes about its DNA repair is due to its Homodimer structure. This allows Interactions with polynucleotide kinase (PNK), DNA polymerase-beta (POLB) and DNA ligase III (LIG3). this protien has tendencies to accumulate around DNA damage possibly making it some form of trans-promoter

  7. Monitoring gene expressions may provide insight into the molecular fingerprints of different diseases such as cancer, cardiovascular diseases and diseases of the central nervous system. Microarray assays large amounts of molecules, scientists monitor the expression level of large amounts of genes, cDNA is used or oligonucleotide chip is used for gene expression analysis. These are spotted on glass slides or nylon. By fabricating custom arrays mechanisms of drug resistance to cancer was investigated. Drug resistance in cancers lead to failure of effective chemotherapy in patients. Some cells resist the chemotherapeutic drug and instead transforms into a resistant tumor. The human breast cancer cell MFV-7 cells are used to compare how the DNA microarray technique is used at the present moment and also its usage for future cancer research purposes. MFV – 7 cells are treated with doxorubicin and some are selected for resistance.
    OMIM Database
    The Cytochrome P450 is a group of enzyme which catalyzes the oxidation of most organic substances. The substrates of these enzymes include metabolic intermediates such as lipids and steroidal hormones as well as xenobiotic substances such as drugs and other toxic chemicals. They are mostly involved in drug metabolism and activation of drugs. The most common reaction catalyzed by CP450 is the monooxygenase reaction. CYP P450 enzymes exist in all of life such as animals, fungi, protists and bacteria. The induction of CYP P450 genes are mediated by specific ligand receptor interaction, at the promoter elements of CYP.

    UNIPROT Database
    When I searched Cytochrome P450 over 100 genes showed up.I chose the one that is in homosapiens. They are located in the Endoplasmic reticulum, Peripheral membrane protein and in Microsome membrane. This gene is expressed in many tissues. It targets the gene CYP1B1.Defects in this gene can cause glaucoma and also be a cause of Peters anomaly which is a congenital defect of the chamber of the anterior eye.

    • Cytochrome p450 is a very interesting group of enzymes and they have a variety of functions . Do you know of other functions and where it located? It is the heart of metabolism.

      • Cytochrome p450 is located in the liver and the small intestines as well. In the liver it metabolizes greatest proportions of drugs.

  8. There was an article published called, Resistance to diverse drugs and ultraviolet light conferred by overexpression of a novel human 26 S proteasome subunit by Spataro V, Toda T, Craig R, Seeger M, Dubiel W, Harris AL, Norbury C. from the Imperial Cancer Research Fund Molecular Oncology Laboratory, University of Oxford Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, United Kingdom. Within this article they have explained that the 26s proteasome is a protein thats influenced by a dependent protein that degrades and targets the ubiquitin pathway.
    -OMIM database:
    http://omim.org/entry/607173
    26s Proteasome is a multiprotein complex that degrades proteins targeted for destruction by the ubiquitin pathway. It is a multidrug resistent cell in humans, with the inability to remove ubiquitin for degradation in vitro it makes it lethal to yeast. Also reported in this database is that this gene seems to depend on zinc which is unlike any other enzymes similar to this one.
    -Uniprot database:
    http://www.uniprot.org/uniprot/P62191
    Within this database 26s proteasome is described as being involved in the ATP-dependent degradation of the ubiquitinated proteins.
    From further research I have found out that this is a very broad gene with a large complex of proteins, it is located within all eukarotic cells specifically the nucleus and the cytoplasms. Its function is to basically degrade or get rid of any unneccessary proteins that can harm or break peptide bonds.

    • What does zinc do for “degrading protein targeted for destruction by the ubiquitin pathway” since 26s proteasome appears to be a zinc-dependent protease?

  9. Even though many cells in our body might contain identical genetic substances, it is hard to tell which are active in those cells. Basically, DNA microarray is a kind of new advanced technology that helps scientists to analysis how these genes within the cells function on the principal of on the principal of base-pairing. Furthermore, researchers are now able to investigate mechanism of drug resistance development in cancer. For example, XRCC1 gene, or X-ray repair complementing defective repair in Chinese hamster cells, is a kind of gene that enhance the repair of drug/enzyme/DNA strand breaks and confer drug resistance in tumor cells. The gene interacts with DNA ligase III, polymerase beta and polymerase to participate in the base excision repair pathway. Also, it plays a role in DNA processing during meiogenesis and recombination in germ cells.
    OMIM-XRCC1
    Based on a study by Bhattacharyya and Banerjee in 2001, XRCC1 is found to be interacted with a truncated POLB that is expressed in primary colorectal and breast tumors and inhibits the normal repair function of wildtype POLB, DNA polymerase beta. They determined that the interaction of the variant POLB and XRCC1 is required for the dominant inhibitory effect.
    UniProt- XRCC1
    Function: Corrects defective DNA strand-break repair and sister chromatid exchange following treatment with ionizing radiation and alkylating agents.
    http://www.uniprot.org/uniprot/P18887

  10. DNA microarray is one of the new technological advancements that aid in the processes of disease studies and pathological functions. With the use of carefully selected cDNA mircoarrays show the differences within different cells and their outcomes after they have been exposed to factors that cause gene expression or mutations. Microoarray technology can be costly or somewhat less costly depending on what type of array is being assayed and where the software used for analysis of the data is used from. Cheaper methods would consist of downloading software from the internet which is at no cost rather that purchasing it from a company however depending on the technology needed cost may not be an option when selecting what it is needed. DNA microarray can be used to further explain the effects of radiation on humans by allowing a rather large number of data to be assayed in a little amount of space. Comparsions can be made in the case study of liver cells being affected by alcohol verses a person who never had a drop of alcohol in their life.
    Uniprot id: Q8VHJ9
    Uniprot.org is a site where genes can be located via an id such as that given above and here you can information like gene name and pathways. The gene name for the id given above is Nesprin 1
    OMIM Nesprin 1
    Nesprin has been found in a study of the gene SYNE1 and nesprin 1 was found to be linked to being a link from the nucleoskeleton to the cytoskeleton. It was also found that that longer nesprin molecules can be found outside the nuclear membrane where as shorter one are found on the inside of the nuclear membrane. The study also found that with the aid of the protien SUN1, nesprin 1 is able to interact with laminas in the inner nuclear membrane.

    • How exactly does Nesprin 1 interact with the inner nuclear membrane meaning which other proteins or molecules does it interact with?

  11. Microarrays have been a huge technological advancement when it comes to studying gene expression for researching diseases and the affects of drugs on a gene. The microarray helps to show which genes are activated and which genes are deactivated that normally would be activated if not exposed to the disease or the drug. But what makes it even more of a technological advancement is that a large amount of genes can be investigated at one time which saves time and helps to show correlations of gene expression to a disease or a certain drug.
    The use of the DNA microarray is shown in the article, Application of DNA microarray in Pharmacogenomics and Toxicogenomics, by Khew- Voon Chin and A.-N. Tony Kong. In this paper genes were uses to demonstrate how microarray can be helpful when trying to research pharmacological and toxicological affects of certain genes in the body. One example was the gene XRCC1, which was found to be involved in the repair of DNA strand breaks after exposure of radiation and alkylating agents.

    OMIM:
    XRCC1 interacts with human polynucleotide kinase along with DNA polymerase- beta and DNA ligase III. XRCC1 with the co association with these three proteins is involved in the repair of single-strand breaks on DNA. XRCC1 does this by stimulating DNA kinase and DNA phosphotase activities of polynucleotide kinase at the site of the damaged DNA, which accelerates the overall repair reaction of the strand.
    http://omim.org/entry/194360?search=XRCC1&highlight=xrcc1

    Unitprot:
    XRCC1 stands for X-ray repair cross-complementing protein 1 and it functions to correct defective DNA strand-break repair and sister chromatid exchange following exposure to radiation and alkylating agents. Since the protein repairs DNA after damage it is usually located in the nucleus.
    http://www.uniprot.org/uniprot/P18887

    • this is a good response. It is very thorough and easy to understand.Good explanation of microarray.

  12. DNA microarrays has allowed scientist to discover many breakthroughs. Microarrays shows large amounts of gene expressions and scientist can analysis these expressions for their research. The paper “Application of DNA microarrays in pharmacogenomics and toxigenomics” by Khew-Voon Chin and A. -N. Tony Kong of the Department of Medicine and Pharmacology in the Cancer Institute of New Jersey, evaluated gene expression from genes that were induced by different compounds. One study they did was on the expression of human breast cancer MCF-7 cells that were treated with doxorubicin. The results showed that the treatment of doxorubin changed the expression on many genes in theMCF-7 cell. One gene that had its expression changed was XRCC1. I searched this gene in the databases UniProt and OMIM to find information on the gene.

    UniProt Database: According to the UniProt database, XRCC1 corrects defective DNA strand-break repair and sister chromid exchange following treatment with ionizing radiation and alkylating agents. Its sequence length is 633AA.

    OMIM: Base on the study by Moser et al. (2007), primary human fibroblasts showed that XRCC1 and LIG3 were essential core components of nucleotide excision repair (NER). Downregulation of LIG3 impaired removal of UV-induced lesions and rejoining of UV-induced nicks in chromosomal DNA. XRCC1-LIG3 and polymerase-delta interacted and co-localized with NER components in a UV-specific manner throughout interphase.

  13. A DNA microarray is a collection of microscopic DNA spots attached to a solid surface, each spot usually represents a particular gene. DNA microarrays are used to measure the expression levels of large numbers of genes simultaneously. The process of microarray analysis involves breaking open a cell to isolate its genetic contents and identifying all the genes that are turned on in a particular cell. In the paper, “Application of DNA Microarrays in Pharmacogenomics and Toxicogenomics” by Khew-Voon Chin and A. -N. Tony Kong, DNA microarrays were used to monitor the gene expression of human breast cancer MCF-7 cells. The analysis was done on cells that were treated with doxorubicin or that were doxorubicin resistant. The results showed that in MCF-7 cells doxorubicin treatment caused temporary changes in the expression of a large number of genes. One of these genes was XRCC1. This gene is involved in the repair of DNA strand breaks following exposure to radiation and alkylating agents. It is believed that the up- regulation or over expression of XRCC1 may enhance the repair of strand breaks and confer drug resistance in tumor cells. I looked up XRCC1 in OMIM and UniProt to gain a greater understand of the gene.
    OMIM: XRCC1 has 17 exons and spans approximately 31.9 k.b. Whitehouse et al. (2001) reported that XRCCI is able to form a multiprotein complexes that repairs single-strand breaks in DNA that are usually induced by reactive oxygen species and ionizing radiation. The complex forms when XRCC1 interacts with PNKP, DNA polymerase, and DNA ligase III.
    http://omim.org/entry/194360?search=XRCC1&highlight=xrcc1
    http://omim.org/entry/606350
    Uniprot Database: This database tells us that XRCC1 corrects defective DNA strand-break and sister chromatid exchange after being treated with ionizing radiation and alkylating agents. In addition, carriers of the polymorphic Gln-399 allele may be greater risk for tobacco and age related DNA damage.
    http://www.uniprot.org/uniprot/P18887

    • I always find genes that respond to carcinogens interesting. I was surprised by the amount of detail on this particular gene you were able extract from the online databases, it just comes to show how these databases are growing.

  14. Nuclear Factor Erythroid 2-Like (NFE2L2) is a family of genes which encode the bZIP. They share many regions with other bZIP, JUN, and FOS families. These sequences from the beta globin locus are used to screen a human myelogenous leukemia cell line, or for the screening of the fetal liver. I found this information using OMIM database.

  15. Microarrays are a fascinating new way to be able to look at genes. It is fairly simple enough that we were able to complete a part of the process in out toxicogenomics lab. It is very cool how so many genes can be represented in such a small space, such as a glass slide. On the microarray are many spots that each contains a gene. I am confused on the part that regards how the genes stick to the microarray. Once you look at the microarray after you finish scanning it, it can come out with green spots, red spots, or yellow spots depending on how much of that gene is expressed in the gene you are testing compared to the control. The gene that I chose to write about was the phase two DME gene (xenobiotic metabolizing enzymes). Using OMIM I found that this gene was tested in mice and the name of it was Nuclear Receptor Subfamily 1. The reactions that this enzyme completes are to help detoxify poisonous substances.

  16. Microarray’s may not be the new thing anymore but they still made and impact in the scientific community. Going to uniprot or omim should suffice as evidence to the contribution that microarrays have given the scientific community. From the paper the gene that I chose to examine is P450
    P450 better known as Cytochrome P450 encodes for the 21- hydroxylase enzyme which plays a pivotal role in adrenal steriodogenisis. The process produces pregnolone which precedes other steroid hormones. The P450 gene itself produces the enzyme that catalyzes hydroxylization and oxygenation reactions.

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