Cyclic carbonate esters
Related Terms
Arylesterase, cardiovascular disease, chlorpyrifos, cyclic carbonate esters, DNA sequencing, ELISA, enzyme linked immunosorbent assay, HDL, high density lipoproteins, insecticides, lactones, LDL oxidation, low density lipoproteins, nerve gas, organophosphates, paraoxonase-1 (PON1) polymorphisms, parathion, Parkinson's disease, PCR, polymerase chain reaction, PON1 status, restriction fragment length polymorphism, RFLP, single nucleotide polymorphisms, SNP.
Background
The paraoxonase (PON1) gene provides instructions (encodes) for the making of the paraoxonase enzyme also known as arylesterase enzyme, which plays a vital role in various bio-chemical (metabolic) reactions in the body. The paraoxonase enzyme was initially identified because of its ability to hydrolyze paraoxon, a toxic metabolite seen in the insecticide (agent that kills insects) parathion. Hydrolysis refers to a chemical reaction of a compound with water and metabolite refers to a substance involved in a biochemical reaction. Paraoxonase enzyme is also associated with complex disorders such as cancer, heart disorders, Parkinson's disease (a progressive nervous disease), and developmental disorders.
Genes, which are made of deoxyribonucleic acid (DNA), are considered the building blocks of life because they provide instructions for all the cells in the body. They are located inside of cells and control an organism's development and function by instructing cells to make new molecules (proteins). Genes are passed from parents to their children. Alleles are two or more alternative forms of a gene that may occur alternatively at a specific site on a chromosome. Chromosomes carry hereditary information in the form of genes. Humans have 22 pairs of chromosomes (autosomes) and a pair of sex chromosomes (X and Y chromosomes). The PON1 gene is located on the long arm of chromosome 7.
A permanent change in a DNA sequence of a gene is called a mutation. Genetic changes or mutations that occur in more than 1% of the general population are called polymorphisms. Some mutations and polymorphisms may influence the risk of the development of certain disorders.
Functions: The PON1 gene encodes for the paraoxonase enzyme and different types of gene polymorphisms determine the amount of the enzyme produced and the different levels of its enzymatic activity. The PON1 enzyme is involved in various biochemical reactions, which may be physiological (reactions occurring as a normal functioning of an organism) and chemical reactions involving substances outside the body (e.g., organophosphorous compounds).
The PON1 enzyme is synthesized in the liver and secreted in blood where it becomes a component of high-density lipoproteins (HDL) and also helps in preserving the HDL particle. HDL is found in the blood and is made of a combination of proteins and fats (lipid). HDL transports the fats in the form of fatty acids and cholesterol from body tissues to the liver for disposal. HDL cholesterol (HDL-C) is also called "good cholesterol," as increased levels of HDL in the blood are associated with a reduced risk of developing heart disease. HDL and PON1 enzyme have a protective effect on the body.
Low density lipoproteins (LDL) are another form of lipoproteins (compounds of proteins and fat), which carry fats in the form of cholesterol and triglycerides in the blood from liver to various tissues of the body. LDL, while transporting cholesterol in the blood, deposit fats or lipids on to the cells of the internal wall of blood vessels by a biochemical process called oxidation. The oxidation process also provokes an inflammatory response-activation of immune system. The repeated deposition of lipids on a long term leads to lipid plaques, also known as atheromas, which in turn blocks or clogs the blood vessels supplying the heart resulting in various heart disorders such as coronary artery disease (CAD). PON1 inhibits the oxidation process of LDL, thereby preventing the risk of the development of heart disorders.
Organophosphorus compounds (organophosphates) are chemical substances that are widely used as insecticides (agent that kills insects) or pesticides (agent that kills pests). Examples include parathion, chlorpyrifos, and nerve gases, which are toxic to the nerves and used in chemical warfare on humans. Chemical warfare refers to the use of lethal or incapacitating agents in military operations or warfare (e.g. the mustard gas used in World War 1 and 2). The PON1 enzyme basically hydrolyzes the paraoxon as well as phenylacetate, which are toxic metabolites of insecticides containing organophosphorus compounds, thereby reducing the effect of these toxic chemicals in the body.
Recent research reports have found that the basic physiological activity of PON1 is the hydrolysis of lactones such as homocysteine thiolactone, homogentisic acid lactone, etc., as well as cyclic carbonate esters (chemical metabolites). Homocysteine thiolactone is a toxic metabolite of homocysteine (an amino acid), which has a negative side effect on protein synthesis that may lead to damage of the blood vessels. A high level of homocysteine is a risk factor for heart diseases. The detoxification of these lactones by PON1 enzyme through hydrolysis has a protective function on the heart.
Methods
The paraoxonase (PON1) gene provides instructions for the making of paraoxonase or arylesterase enzyme, which plays a vital role in various bio-chemical reactions in the body. A permanent variation in a DNA sequence of a gene is called a mutation; mutations that occur in more than 1% of the general population are called polymorphisms. Different types of gene polymorphisms determine the amount of the PON1 enzyme produced and different levels of its enzymatic activity.
PON1 enzyme activity involves the hydrolysis of paraoxon and phenylacetate, which are toxic chemical byproducts (metabolites) of insecticides such as parathion or chlorpyrifos. Other than insecticides, people may get exposed to parathion and chlorpyrifos from acaricides (agent that kills pests such as mites) or avicides (agents killing bird pests) as well as water contaminated from these toxic substances. Hydrolysis refers to a chemical reaction of a compound with water and insecticide refers to an agent that kills insects. PON1 enzyme also plays an essential role in preventing the oxidation of low-density lipoproteins (LDL), thereby reducing the risk of heart diseases. LDLs are a combination of proteins and fats (lipids) that carry fats from the liver to tissues in the body. The oxidation of LDL refers to a biochemical reaction where lipids are deposited on the walls of arteries.
Gene polymorphisms: Some of the polymorphisms associated with PON1 gene are a result of single nucleotide polymorphisms (SNPs). SNPs are deoxyribonucleic acid (DNA) sequence variations that occur when a single nucleotide in the genome sequence is altered. Nucleotides are the building blocks of DNA in a sequence and made of nitrogen bases, sugars, and phosphate. Nitrogen bases are of two types: purines, such as adenine (A) and guanine (G), and pyrimidines, such as cytosine (C) and thymine (T) in DNA. For example, a SNP may change the DNA sequence 'AAGGCTAA' to 'ATGGCTAA.' Here, the adenine (A) base has been substituted with thymine (T). The polymorphisms basically affect the concentration of PON1 enzyme in the blood and thereby may affect the level of enzymatic activity and in turn affect the development of heart disease.
The most common polymorphisms on the PON1 gene are in the coding regions, which include a leucine (L) to methionine (M) transition at position 55 (55L>M) and a glutamine (Q) to arginine (R) transition at position 192 (192Q>R). Leucine, methionine, glutamine, and arginine are amino acids, the building blocks of proteins. Coding region refers to regions of DNA sequence that code for proteins or amino acid sequences, which are triplets of nucleic acid sequence (codon) that specify a single amino acid. The 55L>M polymorphism affects the enzyme concentration in blood and 192Q>R polymorphism is responsible for a substrate specific difference in the hydrolytic activity of the enzyme i.e., increases the enzyme activity. Research data show increased hydrolyzing activity in 192Q>R (arginine substitution), but this varies in different ethnic populations ranging from four to 10 times higher activity. A substrate is a substance on which an enzyme acts and hydrolytic activity refers to a chemical reaction of a compound with water. Thus, these polymorphisms may affect the level of PON1 enzymatic activity and in turn affect its activity to detoxify toxic chemical byproducts such as paraoxon, a very poisonous substance rarely used as an insecticide.
Detection methods: The gene polymorphisms (genotype) may be detected either by restriction fragment length polymorphisms (RFLP) analysis or by DNA sequencing of PON1 gene. The observable characteristics or expression (phenotype) of PON1 enzyme may be detected either by estimating the concentration of PON1 enzyme (enzyme linked immunosorbent assay/ELISA) or estimation of PON1 enzyme activity (by paraoxonase activity) from serum (the fluid portion of blood).
Restriction fragment length polymorphism analysis (RFLP): RFLP analysis is a molecular laboratory technique in which the extracted DNA from a sample is cut (cleaved) into fragments of DNA sequence by the enzymes called restriction endonucleases. These enzymes cleave the DNA sequences at specific sites (recognition restriction sites) resulting in characteristic fragments of DNA of different length and strand orientation.
The fragmented DNA's are put on (run on) gel electrophoresis, which get separated based on their size and electric charges as they migrate through the gel to form distinct bands. The separated DNA fragments are then paired with (hybridized) complementary sequences of DNA called probes. The probes have specific sequences of nucleotides, which are complementary to only specific DNA fragment (target sequence) to which they get hybridized. These special probes are tagged (attached) with a radioactive dye, facilitating the detection by autoradiography. Autoradiography is a technique where the probes are labeled (attached) with radioactive molecules, which on exposure to X-rays may be visualized.
The RFLP analysis technique is a slow and cumbersome procedure requiring a large sample of DNA. However, with the development of polymerase chain reaction (PCR) and its automation, certain limitations of RFLP have been overcome, as PCR may amplify minute quantities of biologic material so as to provide adequate specimens (PCR products) for further analysis. Also, the advancement in detection techniques such as fluorescent imaging has helped the RFLP to become more efficient in the detection of PON1 gene polymorphisms.
DNA sequencing: DNA sequencing is a process in which the exact sequence of nucleotide bases (nucleotides) from a sample DNA is determined. Nucleotides are the building blocks of DNA that are organized in a sequence and made of nitrogenous bases, sugars, and a phosphate group. Usually, Sanger's method (dideoxy or chain termination method) is utilized to detect PON1 gene mutations.
The initial step of DNA sequencing involves the extraction of high quality DNA from the sample of interest, followed by polymerase chain reaction (PCR) in the presence of fluorescent labeled (attached) dideoxynucleotide triphosphate (ddNTP). ddNTPs are synthetic nucleotides that are structurally somewhat different from the regular nucleotides and function as DNA chain terminators (stopper) during the synthesis of a DNA sequence. The end reaction product is a set of DNA sequences differing in length by one nucleotide and the last nucleotide base in each sequence is the unique fluorescent labeled ddNTP.
The reaction products are run on the electrophoresis gel, which separates the DNA fragments based on their size. During this separation, the fluorescent signals are detected by the fluorescent detection systems, thereby identifying the nucleotide base. These fluorescent signals are fed and analyzed by a computer, giving the exact sequence of DNA. The whole process is automated and the resultant DNA sequence is compared with other sequences by various computer programs, thereby spotting the mutations in the sample DNA sequence.
Conventional DNA sequencing is labor intensive, time-consuming, and expensive but, with the development of automated DNA sequencer and newer detection methods, this technique has become very competitive in detecting gene mutations. Other advantages with direct DNA (automated) sequencing are: the complete information it provides in a single experiment such as the type of the mutation, exact location of mutation on the DNA sequence, and the sequence context of each polymorphism associated with the PON1 gene.
Enzyme linked immunosorbent assay: ELISA is a sensitive laboratory technique that uses an enzyme linked to an antigen or antibody as a marker for the detection of a specific protein i.e., the PON1 enzyme or arylesterase. An antigen is a substance (normally a protein, from outside of the body) that makes the body produce another special type of protein (antibody). Antibodies identify and neutralize antigens. Here antibodies against the PON1 enzyme are used to measure the concentration of the PON1 enzyme or arylesterase from serum. The arylesterase activity is the action of the PON1 enzyme to hydrolyze phenyl acetate (arylester), which is a direct indicator of PON1 concentration and is not influenced by the gene polymorphisms.
PON1 enzyme activity: The capacity of blood to hydrolyze paraoxon (paraoxonase activity) is used as a marker for the PON1 enzyme activity. This enzyme activity reflects the combined effects of the 192Q>R polymorphism and the variation in the concentration of the PON1 enzyme. The paraoxonase activity test is based on spectrophotometric analysis, an analytical technique used to determine the intensity of various wavelengths (related to energy of light) of a substance in a spectrum of light.
Research
Currently, research is underway to understand the dietary and pharmacological (drug treatment) means of modifying paraoxonase (PON1) enzyme activity to prevent or reduce the risk of the development of heart diseases. Statins (HMG-CoA reductase inhibitor) are drugs, which indirectly lower the cholesterol in the body through a series of chemical reactions and also have been found to affect PON1 enzymatic activity, its concentration in the blood, and PON1 gene expression, thereby reducing the development of heart disorders. Newer chemicals and drugs are being studied on similar lines to prevent heart diseases.
Newer gene polymorphisms are being identified on the PON1 gene, which has varied gene expressions and thus may affect the levels of the PON1 enzyme in the blood as well as its activity in several biochemical reactions in which it may be involved. This may help to understand the body's response to toxic effects of insecticides based on organophosphorus compounds as well as an independent risk factor for heart diseases. Organophosphorus compounds are chemical substances that are commonly used as insecticides (parathion, chlorpyrifos, etc).
People exposed to nerve agents (organophosphorus compounds) used in non-conventional warfare need to be treated by an injected drug or an enzyme, which may rapidly hydrolyze and destroy the nerve agents or block their action. Currently, PON1 is being studied for this purpose.
Recent research studies have found that PON1 gene polymorphisms and their variable enzyme activity might contribute to the effects of pollutants and other environmental chemicals, which may make certain individuals more vulnerable to cancer, birth defects, and Parkinson's disease (an age-related progressive disease of the central nervous system).
Implications
Cardiovascular diseases: PON1 enzyme is associated with high density lipoproteins (HDL), which plays a vital role in protecting the heart by preventing fat deposits on the walls of blood vessels. By blocking the biochemical process of low density lipoprotein (LDL) oxidation, there is a reduced risk of developing heart disease such as heart attacks (myocardial infarction) and clogging of blood vessels that supply the heart (coronary artery diseases). Cardiovascular diseases refer to diseases of the heart and of the blood vessels that supply the heart. HDL and LDL are compounds of proteins and fats (lipids) that basically carry lipids in the form of cholesterol, fatty acids, and triglycerides in blood circulation. The cardio-protective (protecting the heart) effect of PON1 enzymes may vary due to gene polymorphisms. Hence, individuals with reduced PON1 enzyme activity may be more likely to develop cardiovascular disorders.
Nerve gas agents: The organophosphorus compounds have also been used as nerve gas agents in non-conventional warfare. Persons with elevated levels of PON1 enzyme activity have less toxic effects of these nerve agents in comparison to decreased levels of enzymatic activity due to PON1 gene polymorphisms.
Population distribution: PON1 enzymatic activity may exhibit higher or lower enzyme activity (bimodal distribution) in different population groups. High enzyme activity is characterized by the presence of arginine at position 192 on the PON1 gene sequence, where as the presence of glutamine genetic code at the same position results in reduced PON1 enzyme activity. Arginine and glutamine are amino acids, the building blocks of proteins. The genetic code refers to triplets of a nucleic acid sequence (codon) that specifies a single amino acid. The PON1 enzyme activity in different populations is important to know to prevent the toxic effects of organophosphorus-based compounds and reduce the risk of the development of cardiovascular disorders.
Toxic effects of insecticides: The paraoxonase (PON1) enzyme hydrolyses the paraoxon, which is a toxic chemical byproduct (metabolite) of insecticides containing organophosphorus compounds such as parathion, chlorpyrifos etc., thereby reducing the effect of these toxic chemicals in the body. Hydrolysis refers to a chemical reaction of a compound with water, and insecticide refers to an agent that kills insects. The paraoxonase enzyme activity is of interest especially to farmers who are exposed to these insecticides as well as other persons exposed to this compound occupationally. An individual's ability to detoxify these toxic metabolites depends on the level of PON1 enzymes as well as its activity, which in turn depends on gene polymorphisms on the PON1 gene. Variations in the sequence of a gene detected in more than 1% of general population are called polymorphisms. Hence, persons with reduced PON1 enzyme activity should be cautious when using insecticides based on the organophosphorus compounds so as to prevent or reduce the toxic affects it may cause to the body. It is estimated that 220,000 people die each year from such exposures worldwide. In the United States, around 300,000 poisonings occur from pesticides a year; there are around 25 accidental deaths from pesticides per year.
Limitations
The disadvantage of using DNA sequencing to detect SNPs is that it requires very high quality DNA and is expensive. These disadvantages are being gradually overcome by automation. Another major drawback with this method is that only about 400 base pairs (bp) of sequencing data may be generated by DNA sequencing in a series of experiments, restricting it to smaller fragment sizes, thereby increasing the cost and time.
Future research
The paraoxonase (PON1) gene and its enzymes play an important role in fat metabolism (breakdown) and the prevention of heart disease. A lingering question remains: whether PON1 has any role in the onset (start) of cardiovascular diseases (heart diseases). Further studies are required to understand the role of PON1 in heart disease onset by both genetic variation studies as well as PON1 protein association studies. Genetic variation studies involve newer gene polymorphisms and their effects on enzymatic activities. Body functions and protein association studies involve protein interactions with other proteins or factors. Other factors may be internal (within the body) or external such as environmental agents, drugs, or toxic substances.
The paraoxonase enzyme protects against oxon forms (toxic metabolite or chemical byproduct) of diazinon and chlorpyrifos but may not protect against paraoxon (a toxic metabolite). Diazinon and chlorpyrifos are organophosphorus based insecticides. Further research in this area is required, particularly as increasing attention is given to non-conventional warfare agents such as organophosphorus compounds. More studies also need to be done on the potential susceptibility of PON1 variation to the Gulf War Syndrome since lower levels of PON1 enzyme activity were reported in the affected individuals. Gulf War syndrome refers to illnesses and various symptoms reported by combat veterans of the 1991 Gulf War. Some of the symptoms of Gulf War syndrome include general tiredness, headache, dizziness, memory problems, muscle and joint pain, and skin problems.
Author information
This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).
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