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Structure of cholesterol
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Initial Steps in Cholesterol Synthesis
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Cholesterol synthesis begins with the synthesis of acetoacetyl CoA from two acetyl CoA molecules. A third acetyl CoA molecule is added by the enzyme HMG CoA synthase to form 3 Hydroxy-3-MethylGlutaryl-CoA (HMG CoA). HMG CoA is reduced by the action of HMG CoA reductase to mevalonic acid using NADPH as the hydrogen donor. The end product molecule, cholesterol, acts as a feedback inhibitor of HMG CoA reductase to regulate the intra- and intercellular levels of cholesterol. Lipitor™ is a synthetic lipid lowering agent that works by mimicking the inhibitory effects of cholesterol.
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Cholesterol synthesis 1
Acetyl-CoA is the sole carbon building block for cholesterol synthesis. The intermediate 3-hydroxy- 3-methylglutaryl-CoA (HMG-CoA) sits at an important metabolic branch point: in the cytosol its fate is to the synthesis of polyisoprenes while in the mitochondrion it is the precursor of ketone bodies.
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Cholesterol synthesis 2
The enzyme that catalyzes the reduction of 3-hydroxy-3- methylglutaryl-CoA in the cytosol, 3-hydroxy-3- methylglutaryl-CoA reductase (HMG-CoA reductase), is the major site of regulation the synthesis of cholesterol and other polyisoprenes.
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Cholesterol synthesis 3
In the boxes are the two simple, 5-carbon isoprene structures that are found in ALL sterols, natural rubber, and many other natural products.
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Cholesterol synthesis 4
Polymerization usually occurs with additions of 5 carbons. Infrequently, fusions of larger structures take place, such as the formation of a molecule of squalene from two molecules of farnesyl-pyrophosphate. Squalene is an unusual biological compound, in that it is a pure hydrocarbon (only carbons and hydrogens).
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Cholesterol synthesis 5
The cyclization of squalene to lanosterol is a remarkable series of molecular rearrangements.
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Cholesterol synthesis 6
The loss of the methyl group at carbon-14 as HCOO- (formate, anion of formic acid) yields a substrate for one-carbon metabolism that utilize tetrahydrofolate-bound intermediates.
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Regulation of cholesterol synthesis
Regulation of cholesterol synthesis is focussed on the enzyme 3-hydroxy-3- methylglutaryl-CoA reductase. Hormones alter the level of protein phosphorylation and catalytic activity. Cholesterol appears to act in a feedback loop, either in the short-term (oxidized sterols) or in the long-term, perhaps as a classical steroid hormone. Multiple levels of regulation ensure control of the body's total cholesterol pool. 3-hydroxy-3- methylglutaryl-CoA reductase is also the target of a variety of pharmacologic agents that are used to decrease cholesterol levels in the blood.
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Normal artery
This is a much-simplified cartoon of the cross-section of a major artery presented elsewhere.
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Fatty streak
Fatty streaks appear and disappear with regular frequency throughout adult life.
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Fibrous lesion
Formation of intermediate fibrous lesions is a hallmark of impending pathological consequences. Smooth muscle infiltration and proliferation contribute to contained deposits of fibrous proteins (elastin, collagens), calcium salts, and necrotic debris.
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Large fibrous lesion
The fibrous lesion grows larger and more complex; it occludes blood flow to a greater extent. The advanced complex lesion is more fragile and becomes increasingly more susceptible to fragmentation, a thrombotic event that can quickly produce infarcts in smaller arteries.
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Coronary Atherosclerosis with Hemorrhage
This is coronary atherosclerosis with the complication of hemorrhage into atheromatous plaque, seen here in the center of the photograph. Such hemorrhage may narrow the arterial lumen acutely.
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Coronary heart disease and cholesterol
Increasing TOTAL and LDL cholesterol correlate positively with increased risk for coronary heart disease. Increasing HDL cholesterol correlates negatively; that is, it has a protective effect.
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LDL cholesterol and heart disease
Multiple relationships and pathways have been described between increased LDL cholesterol and the development of advanced atherosclerotic lesions. Cells that participate in this scheme are highlighted. Liver and extrahepatic tissues are also involved, in that they play a critical role in determining the half-life of LDL in the plasma.
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Familial hypercholesterolaemia
Familial hypercholesterolemia is an autosomalDOMINANT disease. Accordingly, even the heterozygote exhibits a clinical phenotype.
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LDL cholesterol clearance
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Low density lipoporotein metabolism
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Oxidised LDL
Oxidized LDL differ from native LDL in that lipids and proteins have been modified by reactive oxygen species (superoxide and hydroxyl radicals and peroxides) that are produced by macrophages and other cells. Contributions of oxidized LDL to the development of atherosclerotic lesions are more pronounced than those of native LDL.
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Lipoprotein (a)
Lipoprotein (a), pronounced lipoprotein little a, is a complex of a normal LDL particle and the apoprotein (a) molecule. Increasing levels of plasma Lp(a) are now identified with increased risk for cardiovascular disease.
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LDL cholesterol and atherosclerosis
Superimposed on the multiple relationships and pathways between increased LDL cholesterol and the development of advanced atherosclerotic lesions are intervention protocols (dietary, exercise, pharmacologic) designed to decrease the risk of cardiovascular disease.
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Cholesterol and cholate
In the metabolism of cholesterol to cholate, a planar, amphipathic molecule that prefers a bilayer environment is transformed into a twisted, highly polarized detergent molecule whose sole purpose in the biological world is to disrupt the bilayers (e.g., cell membranes) it encounters in the digestive process.
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Bile
The enterohepatic circulation links several tissues of the gastrointestinal tract and functions to recycle efficiently (>95%) the bile acid and bile salt pool, such that very little sterol is lost daily. An increase in this loss may be achieved through pharmacological intervention and is one approach to decreasing elevated plasma cholesterol levels.
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Other isoprene derivatives
Polyisoprenes, other than cholesterol and its derivatives, are important in many diverse cellular processes, including farnesyl and geranylgeranyl lipid anchors on membrane proteins and the >100-carbon dolichol for the synthesis of glycoproteins.
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Very Low Density Lipoproteins, LDLs
The dietary intake of both fat and carbohydrate, in excess of the needs of the body, leads to their conversion into triacylglycerols in the liver. These triacylglycerols are packaged into VLDLs and released into the circulation for delivery to the various tissues (primarily muscle and adipose tissue) for storage or production of energy through oxidation. VLDLs are, therefore, the molecules formed to transport endogenously derived triacylglycerols to extra-hepatic tissues. In addition to triacylglycerols, VLDLs contain some cholesterol and cholesteryl esters and the apoproteins, apoB-100, apoC-I, apoC-II, apoC-III and apoE. Like nascent chylomicrons, newly released VLDLs acquire apoCs and apoE from circulating HDLs.
The fatty acid portion of VLDLs is released to adipose tissue and muscle in the same way as for chylomicrons, through the action of lipoprotein lipase. The action of lipoprotein lipase coupled to a loss of certain apoproteins (the apoCs) converts VLDLs to intermediate density lipoproteins (IDLs), also termed VLDL remnants. The apoCs are transferred to HDLs. The predominant remaining proteins are apoB-100 and apoE. Further loss of triacylglycerols converts IDLs to LDLs.
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Intermediate Density Lipoproteins, IDLs
IDLs are formed as triacylglycerols are removed from VLDLs. The fate of IDLs is either conversion to LDLs or direct uptake by the liver. Conversion of IDLs to LDLs occurs as more triacylglycerols are removed. The liver takes up IDLs after they have interacted with the LDL receptor to form a complex, which is endocytosed by the cell. For LDL receptors in the liver to recognize IDLs requires the presence of both apoB-100 and apoE (the LDL receptor is also called the apoB-100/apoE receptor). The importance of apoE in cholesterol uptake by LDL receptors has been demonstrated in transgenic mice lacking functional apoE genes. These mice develop severe atherosclerotic lesions at 10 weeks of age.
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Low Density Lipoproteins, LDLs
The cellular requirement for cholesterol as a membrane component is satisfied in one of two way: either it is synthesized de novo within the cell, or it is supplied from extra-cellular sources, namely, chylomicrons and LDLs. As indicated above, the dietary cholesterol that goes into chylomicrons is supplied to the liver by the interaction of chylomicron remnants with the remnant receptor. In addition, cholesterol synthesized by the liver can be transported to extra-hepatic tissues if packaged in VLDLs. In the circulation VLDLs are converted to LDLs through the action of lipoprotein lipase. LDLs are the primary plasma carriers of cholesterol for delivery to all tissues.
The exclusive apolipoprotein of LDLs is apoB-100. LDLs are taken up by cells via LDL receptor-mediated endocytosis, as described above for IDL uptake. The uptake of LDLs occurs predominantly in liver (75/%), adrenals and adipose tissue. As with IDLs, the interaction of LDLs with LDL receptors requires the presence of apoB-100. The endocytosed membrane vesicles (endosomes) fuse with lysosomes, in which the apoproteins are degraded and the cholesterol esters are hydrolyzed to yield free cholesterol. The cholesterol is then incorporated into the plasma membranes as necessary. Excess intracellular cholesterol is re-esterified by acyl-CoA-cholesterol acyltransferase (ACAT), for intracellular storage. The activity of ACAT is enhanced by the presence of intracellular cholesterol.
Insulin and tri-iodothyronine (T3) increase the binding of LDLs to liver cells, whereas glucocorticoids (e.g., dexamethasone) have the opposite effect. The precise mechanism for these effects is unclear but may be mediated through the regulation of apoB degradation. The effects of insulin and T3 on hepatic LDL binding may explain the hypercholesterolemia and increased risk of athersclerosis that have been shown to be associated with uncontrolled diabetes or hypothyroidism.
An abnormal form of LDL, identified as lipoprotein-X (Lp-X), predominates in the circulation of patients suffering from lecithin-cholesterol acyl transferase (LCAT, see HDL discussion for LCAT function) deficiency or cholestatic liver disease. In both cases there is an elevation in the level of circulating free cholesterol and phospholipids.
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High Density Lipoproteins, HDLs
HDLs are synthesized de novo in the liver and small intestine, as primarily protein-rich disc-shaped particles. These newly formed HDLs are nearly devoid of any cholesterol and cholesteryl esters. The primary apoproteins of HDLs are apoA-I, apoC-I, apoC-II and apoE. In fact, a major function of HDLs is to act as circulating stores of apoC-I, apoC-II and apoE.
HDLs are converted into spherical lipoprotein particles through the accumulation of cholesteryl esters. This accumulation converts nascent HDLs to HDL2 and HDL3. Any free cholesterol present in chylomicron remnants and VLDL remnants (IDLs) can be esterified through the action of the HDL-associated enzyme, lecithin:cholesterol acyltransferase, LCAT. LCAT is synthesized in the liver and so named because it transfers a fatty acid from the C-2 position of lecithin to the C-3-OH of cholesterol, generating a cholesteryl ester and lysolecithin. The activity of LCAT requires interaction with apoA-I, which is found on the surface of HDLs.
Cholesterol-rich HDLs return to the liver, where they are endocytosed. Hepatic uptake of HDLs, or reverse cholesterol transport, may be mediated through an HDL-specific apoA-I receptor or through lipid-lipid interactions. Macrophages also take up HDLs through apoA-I receptor interaction. HDLs can then acquire cholesterol and apoE from the macrophages; cholesterol-enriched HDLs are then secreted from the macrophages. The added apoE in these HDLs leads to an increase in their uptake and catabolism by the liver.
HDLs also acquire cholesterol by extracting it from cell surface membranes. This process has the effect of lowering the level of intracellular cholesterol, since the cholesterol stored within cells as cholesteryl esters will be mobilized to replace the cholesterol removed from the plasma membrane.
The cholesterol esters of HDLs can also be transferred to VLDLs and LDLs through the action of the HDL-associated enzyme, cholesterol ester transfer protein (CETP). This has the added effect of allowing the excess cellular cholesterol to be returned to the liver through the LDL-receptor pathway as well as the HDL-receptor pathway.
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LDL Receptors
LDLs are the principal plasma carriers of cholesterol delivering cholesterol from the liver (via hepatic synthesis of VLDLs) to peripheral tissues, primarily the adrenals and adipose tissue. LDLs also return cholesterol to the liver. The cellular uptake of cholesterol from LDLs occurs following the interaction of LDLs with the LDL receptor (also called the apoB-100/apoE receptor). The sole apoprotein present in LDLs is apoB-100, which is required for interaction with the LDL receptor.
The LDL receptor is a polypeptide of 839 amino acids that spans the plasma membrane. An extracellular domain is responsible for apoB-100/apoE binding. The intracellular domain is responsible for the clustering of LDL receptors into regions of the plasma membrane termed coated pits. Once LDL binds the receptor, the complexes are rapidly internalized (endocytosed). ATP-dependent proton pumps lower the pH in the endosomes, which results in dissociation of the LDL from the receptor. The portion of the endosomal membranes harboring the receptor are then recycled to the plasma membrane and the LDL-containing endosomes fuse with lysosomes. Acid hydrolases of the lysosomes degrade the apoproteins and release free fatty acids and cholesterol. As indicated above, the free cholesterol is either incorporated into plasma membranes or esterified (by ACAT) and stored within the cell.
The level of intracellular cholesterol is regulated through cholesterol-induced suppression of LDL receptor synthesis and cholesterol-induced inhibition of cholesterol synthesis. The increased level of intracellular cholesterol that results from LDL uptake has the additional effect of activating ACAT, thereby allowing the storage of excess cholesterol within cells. However, the effect of cholesterol-induced suppression of LDL receptor synthesis is a decrease in the rate at which LDLs and IDLs are removed from the serum. This can lead to excess circulating levels of cholesterol and cholesteryl esters when the dietary intake of fat and cholesterol exceeds the needs of the body. The excess cholesterol tends to be deposited in the skin, tendons and (more gravely) within the arteries, leading to atherosclerosis.
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Apo-B, E receptor
Like many proteins the apo-B,E receptor contains within its primary structure a number of well- defined domains. The functions of most of these domains have not yet been elucidated.
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Clinical Significances of Lipoprotein Metabolism
Fortunately, few individuals carry the inherited defects in lipoprotein metabolism that lead to hyper- or hypolipoproteinemias. Persons suffering from diabetes mellitus, hypothyroidism and kidney disease often exhibit abnormal lipoprotein metabolism as a result of secondary effects of their disorders. For example, because lipoprotein lipase (LPL) synthesis is regulated by insulin, LPL deficiencies leading to Type I hyperlipoproteinemia may occur as a secondary outcome of diabetes mellitus. Additionally, insulin and thyroid hormones positively affect hepatic LDL-receptor interactions; therefore, the hypercholesterolemia and increased risk of athersclerosis associated with uncontrolled diabetes or hypothyroidism is likely due to decreased hepatic LDL uptake and metabolism.
Of the many disorders of lipoprotein metabolism, familial hypercholesterolemia (FH) may be the most prevalent in the general population. Heterozygosity at the FH locus occurs in 1:500 individuals, whereas, homozygosity is observed in 1:1,000,000 individuals. FH is an inherited disorder comprising four different classes of mutation in the LDL receptor gene. The class 1 defect (the most common) results in a complete loss of receptor synthesis. The class 2 defect results in the synthesis of a receptor protein that is not properly processed in the Golgi apparatus and therefore is not transported to the plasma membrane. The class 3 defect results in an LDL receptor that is incapable of binding LDLs. The class 4 defect results in receptors that bind LDLs but do not cluster in coated pits and are, therefore, not internalized.
FH sufferers may be either heterozygous or homologous for a particular mutation in the receptor gene. Homozygotes exhibit grossly elevated serum cholesterol (primarily in LDLs). The elevated levels of LDLs result in their phagocytosis by macrophages. These lipid-laden phagocytic cells tend to deposit within the skin and tendons, leading to xanthomas. A greater complication results from cholesterol deposition within the arteries, leading to atherosclerosis, the major contributing factor of nearly all cardiovascular diseases.
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Hyperlipoproteinemias
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Hypolipoproteinemias
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Pharmacologic Intervention
Drug treatment to lower plasma lipoproteins and/or cholesterol is primarily aimed at reducing the risk of athersclerosis and subsequent coronary artery disease that exists in patients with elevated circulating lipids. Drug therapy usually is considered as an option only if non-pharmacologic interventions (altered diet and exercise) have failed to lower plasma lipids
Mevinolin, Mevastatin, Lovastatin: These drugs are fungal HMG-CoA reductase inhibitors. The net result of treatment is an increased cellular uptake of LDLs, since the intracellular synthesis of cholesterol is inhibited and cells are therefore dependent on extracellular sources of cholesterol. However, since mevalonate (the product of the HMG-CoA reductase reaction) is required for the synthesis of other important isoprenoid compounds besides cholesterol, long-term treatments carry some risk of toxicity.
Nicotinic acid: Nicotinic acid reduces the plasma levels of both VLDLs and LDLs by inhibiting hepatic VLDL secretion, as well as suppressing the flux of FFA release from adipose tissue by inhibiting lipolysis. Because of its ability to cause large reductions in circulating levels of cholesterol, nicotinic acid is used to treat Type II, III, IV and V hyperlipoproteinemias.
Clofibrate, Gemfibrozil, Fenofibrate: These compounds are derivatives of fibric acid and promote rapid VLDL turnover by activating lipoprotein lipase. They also induce the diversion of hepatic free fatty acids from esterification reactions to those of oxidation, thereby decreasing the liver's secretion of triacylglycerol- and cholesterol-rich VLDLs.
Probucol: Probucol increases the rate of LDL metabolism and may block the intestinal transport of cholesterol. The net result is a significant reduction in plasma cholesterol levels.
Cholestyramine or colestipol (resins): These compounds are nonabsorbable resins that bind bile acids which are then not reabsorbed by the liver but excreted. The drop in hepatic reabsorption of bile acids releases a feedback inhibitory mechanism that had been inhibiting bile acid synthesis. As a result, a greater amount of cholesterol is converted to bile acids to maintain a steady level in circulation. Additionally, the synthesis of LDL receptors increases to allow increased cholesterol uptake for bile acid synthesis, and the overall effect is a reduction in plasma cholesterol. (This treatment is ineffective in homozygous FH patients, since they are completely deficient in LDL receptors.
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Cytochrome P-450 enzymes
Cytochrome P-450 enzymes form a large family of membrane-bound, heme-containing proteins. Some are expressed constitutively in tissues, while others are induced by their substrate(s). Each is linked to an electron transport system that provides electrons and protons for the overall reaction.
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Steroid hormone synthesis 1
In this example of the tissue-specific synthesis of one class of steroid hormones, several hydroxylations take place. Each is catalyzed by a cytochrome P-450 enzyme.
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Steroid hormone synthesis 2
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Rapid Identification of Potential Carcinogens using the Tg.AC (v-Ha-ras) and the Heterozygous p53-deficient (+/-) Transgenic Mouse Models
Cancer Biology Group, Laboratory of Environmental Carcinogenesis and Mutagenesis, and National Toxicology Program, NIEHS, NIH, Research Triangle Park, NC 27709
Public Health Significance
Environmental factors and exposure to natural and synthetic chemicals, interacting through behavioral and genetic components, are known or suspected of being major determinants of cancer in humans, a multigenic and multifactorial process. Background and/or induced mutations and/or altered gene expression are believed by biomedical scientists to play critical roles in the induction of cancer, and, most likely, many other somatic diseases. The relationship between our environment and the mutagenesis and carcinogenesis of the human genome is still poorly understood. Most important, retrospective identification of relationships between exposure to hazardous agents by epidemiological research methods and environmental disease requires many years and evidence of human morbidity and death to establish. Prospective identification of chemical hazards and levels of risk is required to minimize or prevent exposure. Understanding the potential multiple pathways that our environment may induce or influence cancer and environmental disease is also important so that means of intervention and/or treatments of cancer and environmental disease may be developed.
Research Initiatives
At the present time, prospective identification of potential chemical carcinogens requires 5 to 7 years of resource intense short and long term toxicology and carcinogenesis investigation. This delays both the determination of toxic and carcinogenic potential and risk assessment. In addition, investigation of potential mechanisms for cancer induction and relevance to human cancer are difficult and, most often, lacking. We are investigating the potential use of transgenic mouse models for mutagenesis and carcinogenesis for rapid identification of environmental mutagens/carcinogens and mechanisms of tissue specific molecular toxic effects associated with the induction of experimentally induced cancers in rodents as surrogates for humans.
Principal Hypothesis: Transgenic mice, with a specific genetic alteration (inducible protooncogene and/or inactivation of a tumor suppressor gene) critical to tumorigenesis, but insufficient by itself to induce cancer, are candidate test species for rapid cancer bioassays. Exposure of such mice to transspecies carcinogens will result in the rapid induction (decreased latency) of tumors.
Background:
Tg.AC mice carry a v-Ha-ras oncogene fused to the promoter of the -globin gene. The v-Ha-ras transgene has point mutations at codons 12 and 59 and the site of integration of the transgene confers on these mice the characteristic of genetically initiated skin as a target for tumorigenesis in the context of the well known, intensively studied, mouse-skin tumorigenesis model. An important consideration of the Tg.AC mouse model is that the transgene is not constitutively expressed in the skin and the untreated skin appears normal when compared to the skin of the wild type FVB/N parent strain. In addition, the spontaneous incidence of skin papillomas in the dorsal skin of untreated mice is very low to zero. Dermal application of carcinogens, but not non-carcinogens, results in activation of the transgene and induction of benign skin tumors (see the list of publications below). The induction of the transgene in the skin and associated tumorigenesis acts as a reporter phenotype for the rapid identification of potential carcinogens by any potential route of application in conventional mouse models. The mechanistic basis for this reporter phenotype in this transgenic mouse is under investigation.
Heterozygous p53 (+/-) Deficient Mice with only a single wild type p53 allele provide a distinct target for mutagens and are analogous to humans at risk due to heritable forms of cancer, e.g., the Li-Fraumeni syndrome (8). The reduction in p53 gene dosage by this "germline first hit" increases both (a) the probability that a second mutagenic event will cause either loss of p53 tumor suppressor function or a gain of transforming activity by requiring (at minimum) only a single mutation (in the remaining functional p53 allele) and/or (b) create permissive conditions (i.e. genomic instability) for clonal expansion of cells harboring mutation(s) in other genes critical to tumorigenesis. Mice nullizygous for p53 (-/-) genes are viable (but have a high rate of early spontaneous tumors at sites apparently determined by the strain's genetic background). In the heterozygous state (one wildtype and one null allele), mice have a low background tumor incidence for up to 12 months of age, thus, allowing a sufficient period for testing free from strain specific background tumor incidence. Recent evidence suggest that the induction of cancer may indirectly or directly involve the p53 tumor suppressor gene. This suggests that the model should have a broad range of chemically induced tumor susceptibility without being overtly sensitive.
Approach: Research efforts are presently divided along two lines of investigation:
validation of the Tg.AC (v-Ha-ras) and p53-deficient (+/-) mouse models for rapid identification of potential mutagenic carcinogens by prospective testing of NTP chemical in progress for two year toxicology and carcinogenesis studies and molecular biology studies designed to determine the role and function of the Tg.AC transgene in skin carcinogenesis and the role of the p53 tumor suppressor gene in mutagenesis and carcinogenesis and identification of critical genes involved in mouse carcinogenesis for comparison to human cancers of the same histogenetic type. Initial studies on the validity of using these two transgenic mouse models to identify potential carcinogens have focused on replicating two-year NCI/NTP cancer bioassays using a diverse group of mutagenic and nonmutagenic carcinogens, as well as a mutagenic noncarcinogen in rapid cancer bioassays. Concordance was very high between positive responses for both genotoxic and nongenotoxic carcinogens in the Tg.AC mouse and for mutagenic carcinogens in the heterozygous p53 deficient mouse and the two year NTP cancer bioassays.
At present two different groups of chemicals are currently under study in this mouse model, as well as the Tg.AC mouse model. The first group chosen by NIEHS/NTP scientists contain both human carcinogens as well as rodent mutagenic and nonmutagenic carcinogens to determine the range of potential responses in these two transgenic mouse lines. The second group represents another approach to validation of the model: the prospective testing of chemicals currently being tested in the NTP cancer bioassay. Prospective analysis provides the opportunity to remove the potential bias of selection of chemicals for validation. A high correspondence between this short-term cancer bioassay and the long-term bioassay (of both groups) would provide assurance that mutagenic carcinogens, may be identified using this mouse model. We anticipate that these studies will be completed by the beginning of the 3rd Quarter of FY97. .
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