Pharmacogenetics in American Indian populations: analysis of CYP2D6, CYP3A4, CYP3A5, and CYP2C9 in the Confederated Salish and Kootenai Tribes.

OBJECTIVES: Cytochrome P450 enzymes play a dominant role in drug elimination and variation in these genes is a major source of interindividual differences in drug response. Little is known, however, about pharmacogenetic variation in American Indian and Alaska Native (AI/AN) populations. We have developed a partnership with the Confederated Salish and Kootenai Tribes (CSKT) in northwestern Montana to address this knowledge gap. METHODS: We resequenced CYP2D6 in 187 CSKT individuals and CYP3A4, CYP3A5, and CYP2C9 in 94 CSKT individuals. RESULTS: We identified 67 variants in CYP2D6, 15 in CYP3A4, 10 in CYP3A5, and 41 in CYP2C9. The most common CYP2D6 alleles were CYP2D6*4 and *41 (20.86 and 11.23%, respectively). CYP2D6*3, *5, *6, *9, *10, *17, *28, *33, *35, *49, *1xN, *2xN, and *4xN frequencies were less than 2%. CYP3A5*3, CYP3A4*1G, and *1B were detected with frequencies of 92.47, 26.81, and 2.20%, respectively. Allelic variation in CYP2C9 was low: CYP2C9*2 (5.17%) and *3 (2.69%). In general, allele frequencies in CYP2D6, CYP2C9, and CYP3A5 were similar to those observed in European Americans. There was, however, a marked divergence in CYP3A4 for the CYP3A4*1G allele. We also observed low levels of linkage between CYP3A4*1G and CYP3A5*1 in the CSKT. The combination of nonfunctional CYP3A5*3 and putative reduced function CYP3A4*1G alleles may predict diminished clearance of CYP3A substrates. CONCLUSION: These results highlight the importance of carrying out pharmacogenomic research in AI/AN populations and show that extrapolation from other populations is not appropriate. This information could help optimize drug therapy for the CSKT population.

Modified low density lipoproteins binding requires a lysine cluster region in the murine macrophage scavenger receptor class A type II.

Atherosclerosis is a consequence of lipid deposition and foam cell formation in the arterial wall. Macrophage scavenger receptor A II is involved in the uptake of modified low density lipoproteins. It contains an extracellular conserved lysine cluster which has been proposed to form a positively charged groove that interacts with acetylated low density lipoproteins (AcLDL). This study evaluated the role of the murine SRA-II and a lysine mutated SRA-II on AcLDL uptake. Fluorescence labeled AcLDL uptake was quantified using a Laser Scan Cytometer. A significant increase in fluorescence uptake was found in the cells transfected with SRA-II versus those with empty vector. Cells expressing the lysine mutated SRA-II also demonstrated a significant decrease in their uptake of AcLDL. This data supports the concept that the conserved lysine cluster in murine SRA-II is the binding region for AcLDL or contributes to the trimeric structure of SRA-II necessary for AcLDL binding.

Differences in gene expression profiles from asbestos-treated SPARC-null and wild-type mouse lungs.

The role of SPARC in the in vivo lung response to crocidolite asbestos was addressed by instillation of crocidolite asbestos in a series of wild-type or SPARC-null mice. Animals were sacrificed at one week, one month, and three months post-instillation to assess the impact of SPARC on multiple stages in the development of fibrosis. RNA was harvested from 10 animals/time point, pooled, and used to probe a mouse array containing approximately 10,000 probes. Gene expression data were analyzed for fold change, and for broader functional group alterations. As expected, the one-week time point displayed alterations in genes involved in immune recognition, energy utilization, and growth factor production. Later time points showed expression alterations for genes involved in protein degradation, Wnt receptor signaling, membrane protein activity, and transport. Molecules in the Wnt pathway have been implicated in bone growth, mediation of fibroblast activity, and have been directly linked to SPARC regulation.

Potential surrogate markers for gamma-hydroxybutyrate administration may extend the detection window from 12 to 48 hours.

The exogenous administration of gamma-hydroxybutyrate (GHB) as a drug of abuse, and especially in date rape sexual assaults, has recently increased. Chromatographic techniques are used to detect GHB in blood or urine, with a window of detection limited to 12 h. This brief window makes the proof of administration problematic in most rape cases. This study is aimed to extend the window of detection through surrogate markers of GHB administration. Microarray technology is used in a DBA/2J mouse model to detect gene expression changes in peripheral blood after GHB exposure at times as long as 96 h post exposure. This study focuses on two of the most significantly altered transcripts, epiregulin and phosphoprotein enriched in astrocytes 15 (Pea-15). Both genes have increased the ribonucleic acid expression (8.5- and 4.6-fold upregulation at 96 h, respectively) in GHB-dosed mice (1 g/kg) as compared with the control. To confirm these results at the protein level, an intracellular flow cytometric assay is developed to detect protein level changes in the peripheral blood of both these potential biomarkers after GHB exposure. These results suggest that after further development, epiregulin and Pea-15 may prove to be significant surrogate markers in the indirect detection of GHB administration.

The role of SV40 in malignant mesothelioma and other human malignancies.

SV40 is a DNA tumor virus thrust upon human populations primarily as a contaminant in various vaccine preparations. Some estimates suggest that millions of people are currently infected with the virus. The virus causes primary brain tumors, bone tumors, lymphomas, and mesotheliomas when injected into some rodent models. It has also been detected in a similar spectrum of human tumors. However, epidemiological studies have failed to conclusively demonstrate a higher incidence of disease in affected populations. To date, over 60 reports from 49 different laboratories have shown SV40 sequences in tissues from human cancer patients. Six studies, however, have failed to detect evidence of virus in similar tissues. Some have suggested that SV40 may act as a cocarcinogen with asbestos to cause mesothelioma formation, or that it may be responsible for the 10-20% of mesotheliomas with no reported history of asbestos exposure. This report briefly covers the historical evidence for SV40 carcinogenesis and then covers experiments now underway to better understand the role of SV40 in human mesotheliomas.

Partnership with the Confederated Salish and Kootenai Tribes: Establishing an Advisory Committee for Pharmacogenetic Research.

BACKGROUND: Inclusion of American Indian and Alaska Native (AI/AN) populations in pharmacogenetic research is key if the benefits of pharmacogenetic testing are to reach these communities. Community-based participatory research (CBPR) offers a model to engage these communities in pharmacogenetics. OBJECTIVES: An academic-community partnership between the University of Montana (UM) and the Confederated Salish and Kootenai Tribes (CSKT) was established to engage the community as partners and advisors in pharmacogenetic research. METHODS: A community advisory committee, the Community Pharmacogenetics Advisory Council (CPAC), was established to ensure community involvement in the research process. To promote bidirectional learning, researchers gave workshops and presentations about pharmacogenetic research to increase research capacity and CPAC members trained researchers in cultural competencies. As part of our commitment to a sustainable relationship, we conducted a self-assessment of the partnership, which included surveys and interviews with CPAC members and researchers. RESULTS: Academic and community participants agree that the partnership has promoted a bidirectional exchange of knowledge. Interviews showed positive feedback from the perspectives of both the CPAC and researchers. CPAC members discussed their trust in and support of the partnership, as well as having learned more about research processes and pharmacogenetics. Researchers discussed their appreciation of CPAC involvement in the project and guidance the group provided in understanding the CSKT community and culture. DISCUSSION: We have created an academic-community partnership to ensure CSKT community input and to share decision making about pharmacogenetic research. Our CBPR approach may be a model for engaging AI/AN people, and other underserved populations, in genetic research.

Quantitative and qualitative methods using fluorescence microscopy for the study of modified low density lipoproteins uptake.

Atherosclerosis and heart disease are the main cause of death in United States. The development of atherosclerosis includes lipid deposition and foam cell formation in the artery wall. Scavenger Receptors A-I and II (SRA-I/II) have an important role of in foam cell formation and atherogenesis. Most of the SRA-I/II studies had been performed using Iodine-125-radiolabeled modified low-density lipoprotein. This report attempts to validate the use of fluorescence microscopy techniques as an alternative to obtain qualitative and quantitative information of the uptake of fluorescence-labeled acetylated low-density lipoprotein (AcLDL) in adherent CHO cells expressing SRA-I/II. After verifying the protein expression of SRA-I and II, uptake was quantified using a Laser Scan Cytometer, and images of cells containing fluorescent AcLDL were obtained. A significant increase in fluorescence was found in the cells transfected with SRA-I/II vs. those with empty vector. When SRA-I/II competitive ligands were used, the uptake of AcLDL was significantly decreased. In conclusion, the use of fluorescence microscopy techniques in obtaining qualitative and quantitative information of the uptake of fluorescence-labeled AcLDL by adherent cells, such as CHO cells, is an alternative to the traditional use of radiolabeled iodine.

Suppression of the mouse double minute 4 gene causes changes in cell cycle control in a human mesothelial cell line responsive to ultraviolet radiation exposure.

The TP53 tumor suppressor gene is the most frequently inactivated gene in human cancer identified to date. However, TP53 mutations are rare in human mesotheliomas, as well as in many other types of cancer, suggesting that aberrant TP53 function may be due to alterations in its regulatory pathways. Mouse double minute 4 (MDM4) has been shown to be a key regulator of TP53 activity, both independently as well as in concert with its structural homolog, Mouse Double Minute 2 (MDM2). The purpose of this study was to characterize the effects of MDM4 suppression on TP53 and other proteins involved in cell cycle control before and after ultraviolet (UV) exposure in MeT5a cells, a nonmalignant human mesothelial line. Short hairpin RNA (shRNA) was used to investigate the impact of MDM4 on TP53 function and cellular transcription. Suppression of MDM4 was confirmed by Western blot. MDM4 suppressed cells were analyzed for cell cycle changes with and without exposure to UV. Changes in cell growth as well as differences in the regulation of direct transcriptional targets of TP53, CDKN1A (cyclin-dependent kinase 1alpha, p21) and BAX, suggest a shift from cell cycle arrest to apoptosis upon increasing UV exposure. These results demonstrate the importance of MDM4in cell cycle regulation as well as a possible role inthe pathogenesis of mesothelioma-type cancers.

Gene expression changes after exposure to six-mix in a mouse model.

The exposure of Libby MT residents to amphibole-contaminated vermiculite is well known. To explore the gene-environment interactions in the development of asbestos-related diseases (ARD), a mouse model of asbestos exposure using Six-mix (a combination of amphibole fibers gathered from six sites at the Libby vermiculite mine), crocidolite asbestos, or saline as a negative control was used to determine both gene expression responses by using mouse 10,000 oligonucleotide array and to visualize these changes histologically. Mice were sacrificed and whole lungs harvested for histology and microarray analysis six months following exposure via intratracheal instillation. Using an arbitrary cutoff of 1.25-fold change, genes whose RNA expression levels were specifically altered in response to the different amphibole exposures were grouped into categories by a gene ontology analysis program, GoMiner. Our hypothesis was that assessment of asbestos-responsive genes would provide a better understanding of response mechanisms. These experiments have provided new candidates for genes involved in the asbestos response pathways.

Effects of rottlerin on silica-exacerbated systemic autoimmune disease in New Zealand mixed mice.

Environmental crystalline silica exposure has been associated with formation of autoantibodies and development of systemic autoimmune disease, but the mechanisms leading to these events are unknown. Silica exposure in autoimmune-prone New Zealand mixed (NZM) mice results in a significant exacerbation of systemic autoimmunity as measured by increases in autoantibodies and glomerulonephritis. Previous studies have suggested that silica-induced apoptosis of alveolar macrophages (AM) contributes to the generation of the autoantibodies and disease. Rottlerin has been reported to inhibit apoptosis in many cell types, possibly through direct or indirect effects on PKCdelta. In this study, rottlerin reduced silica-induced apoptosis in bone marrow-derived macrophages as measured by DNA fragmentation. In NZM mice, RNA and protein levels of PKCdelta were significantly elevated in AM 14 wk after silica exposure. Therefore, rottlerin was used to reduce apoptosis of AM and evaluate the progress of silica-exacerbated systemic autoimmune disease. Fourteen weeks after silica exposure, NZM mice had increased levels of anti-histone autoantibodies, high proteinuria, and glomerulonephritis. However, silica-instilled mice that also received weekly instillations of rottlerin had significantly lower levels of proteinuria, anti-histone autoantibodies, complement C3, and IgG deposition within the kidney. Weekly instillations of rottlerin in silica-instilled NZM mice also inhibited the upregulation of PKCdelta in AM. Together, these data demonstrate that in vivo treatment with rottlerin significantly decreased the exacerbation of autoimmunity by silica exposure.

Silica, apoptosis, and autoimmunity.

Relatively little is known regarding mechanisms of environmental exposures in the development of autoimmune disease. However, several environmental agents are implicated in triggering or accelerating systemic autoimmune disease, including mercury, iodine, vinyl chloride, certain pharmaceuticals, and crystalline silica. There is increasing epidemiological evidence supporting the hypothesis that occupational silica exposure is associated with a variety of systemic autoimmune diseases, including scleroderma (SSc), rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), glomerulonephritis (GN) and small vessel vasculitis (SVV). However, there have been few mechanistic studies examining silica exposure and autoimmune disease initiation and progression. This review summarizes human epidemiology data linking silica exposure with systemic autoimmune disease, but focuses on possible mechanisms by which silica can lead to the development and progression of autoimmunity.