BIOMONITORING

Biomonitoring
In human : direct measurement of levels of chemical substances in blood, urine, breast-milk or saliva; and such other tissues as bone, teeth, skin, hair and nails Wildlife and other species may include additional tissues such as muscle (for example fish fillets), liver, fat, eggs, and reproductive tissues

Contd
biomonitoring has become the standard for assessing people's exposure to toxic substances for responding to serious environmental public health problems

Biotransformation
is the process whereby a substance is changed from one chemical to another (transformed) by a chemical reaction within the body

Contd
vital to survival in that it transforms absorbed nutrients (food, oxygen, etc.) into substances required for normal body functions it is a metabolite that is therapeutic and not the absorbed drug an important defense mechanism in that toxic xenobiotics and body wastes are converted into less harmful substances and substances that can be excreted from the body.

Contd
Lipophilic toxicant, non-polar, and low molecular weight are readily absorbed through the cell membranes of the skin, gastrointestinal (GI) tract, and lung. Lipophilic toxicants are hard for the body to eliminate and can accumulate to hazardous levels. Most lipophilic toxicants can be transformed into hydrophilic that are easier for the body to eliminate than lipophilic substances

Contd
The human body has a well-developed capacity to biotransform most xenobiotics as well as body wastes Hemoglobin the normal destruction of red blood cells biotransformed to bilirubin toxic to the brain of newborns irreversible brain injury Biotransformation of the lipophilic bilirubin molecule in the liver results in the production of water-soluble (hydrophilic) metabolites excreted into bile and eliminated via the feces.

Contd
The biotransformation process is not perfect Biotransformation results in metabolites of lower toxicity detoxification Metabolites are more toxic than the parent substance bioactivation. An unusually reactive metabolite that may interact with cellular macromolecules (e.g., DNA) cancer or birth defects Biotransformation of vinyl chloride to vinyl chloride epoxide, which covalently binds to DNA and RNA, a step leading to cancer of the liver.

Chemical Reaction
Most of these chemical reactions occur at significant rates only because specific proteins, known as enzymes, are present to catalyze them, that is, accelerate the reaction. A catalyst a substance that can accelerate a chemical reaction of another substance without itself undergoing a permanent chemical change.

Contd
Persons that have phenylketonuria (PKU), a genetic condition in which the enzyme that can biotransform phenylalanine to tyrosine (another amino acid) is defective phenylalanine can build up in the body severe mental retardation.

Contd
Enzymatic reactions are not always simple biochemical reactions Some enzymes require the presence of cofactors or co-enzymes in addition to the substrate (the substance to be catalyzed) before their catalytic activity can be exerted Co-factors exist as a normal component in most cells and are frequently involved in common reactions to convert nutrients into energy (vitamins are an example of co-factors)

Contd
Most biotransforming enzymes are high molecular weight proteins, composed of chains of amino acids linked together by peptide bonds Most enzymes will catalyze the reaction of only a few substrates, meaning that they have high "specificity Specificity is a function of the enzyme's structure and its catalytic sites, referred to as the "lock and key" relationship

Three main type of specificity

The biotransformation of ethyl alcohol to acetaldehyde

ADH = alcohol dehydrogenase

Phase I Reaction
Phase I reactions are 1. Oxidation, 2. Reduction, and 3. Hydrolysis

Oxidation
Chemical reaction in which a substrate loses electrons Addition of oxygen was the first of these reactions discovered The simplest type of oxidation reaction is dehydrogenation, that is the removal of hydrogen from the molecule Another example of oxidation is electron transfer

Types of oxidizing reactions

Oxidizing Reactions
alcohol dehydrogenation aldehyde dehydrogenation alkyl/acyclic hydroxylation aromatic hydroxylation deamination desulfuration N-dealkylation N-hydroxylation N-oxidation O-dealkylation sulphoxidation

Reduction
Chemical reaction in which the substrate gains electron Most likely to occur with xenobiotics in which oxygen content is low Can occur across nitrogen-nitrogen double bonds (azo reduction) or on nitro groups (NO2). Some chemicals such as carbon tetrachloride can be reduced to free radicals, which are quite reactive with biological tissues Result in activation of a xenobiotic rather than detoxification

Reduction reaction in the nitro group

Reducing Reactions
azo reduction dehalogenation disulfide reduction nitro reduction N-oxide reduction sulfoxide reduction

Hydrolysis
Chemical reaction in which the addition of water splits the toxicant into two fragments or smaller molecules The hydroxyl group (OH-) is incorporated into one fragment and the hydrogen atom is incorporated into the other Larger chemicals such as esters, amines, hydrazines, and carbamates are generally biotransformed by hydrolysis

Procaine (local anesthetic) hydrolysis

Phase II Reactions
Are conjugation reactions, that is, a molecule normally present in the body is added to the reactive site of the Phase I metabolite The result is a conjugated metabolite that is more water-soluble than the original xenobiotic or Phase I metabolite Usually the Phase II metabolite is quite hydrophilic and can be readily eliminated from the body.

Contd
The primary Phase II reactions are: glucuronide conjugation - most important reaction sulfate conjugation - important reaction acetylation amino acid conjugation glutathione conjugation methylation

Glucuronide conjugation
One of the most important and common Phase II reactions One of the most popular molecules added directly to the toxicant or its phase I metabolite is glucuronic acid, a molecule derived from glucose, a common carbohydrate (sugar) that is the primary source of energy for cells The sites of glucuronidation reactions are substrates having an oxygen, nitrogen or sulfur bond Includes a wide array of xenobiotics as well as endogenous substances, such as bilirubin, steroid hormones and thyroid hormones

Glucuronide conjugation
Glucuronidation is a high-capacity pathway for xenobiotic conjugation Glucuronide conjugation usually decreases toxicity, although there are some notable exceptions, for example, the production of carcinogenic substances The glucuronide conjugates are generally quite hydrophilic and are excreted by the kidney or bile, depending on the size of the conjugate

Glucuronide conjugation of aniline

Sulfate conjugation
another important Phase II reaction that occurs with many xenobiotics In general, sulfation decreases the toxicity of xenobiotics Unlike glucuronic acid conjugates that are often eliminated in the bile, the highly polar sulfate conjugates are readily secreted in the urine In general, sulfation is a low-capacity pathway for xenobiotic conjugation Often glucuronidation or sulfation can conjugate the same xenobiotics.

The major transformation reactions for xenobiotics

Biotransformation Sites
Biotransforming enzymes are widely distributed throughout the body The liver is the primary biotransforming organ due to its large size and high concentration of biotransforming enzymes, it is also potentially quite vulnerable to the toxic action of a xenobiotic that is activated to a more toxic compound The kidneys and lungs are next with 10-30% of the liver's capacity Low capacity exists in the skin, intestines, testes, and placenta.

Contd
The liver receives blood directly from the gastrointestinal tract via the portal vein "first pass" phenomena Blood leaving the liver is eventually distributed to all other areas of the body the liver may have removed most of the potentially toxic chemical, or some toxic metabolites are in high concentration in the liver.

Contd
Within the liver cell, the primary subcellular components that contain the transforming enzymes are the microsomes (small vesicles) of the endoplasmic reticulum and the soluble fraction of the cytoplasm (cytosol) The mitochondria, nuclei, and lysosomes contain a small level of transforming activity

Contd
Microsomal enzymes are associated with most Phase I reactions Glucuronidation enzymes are contained in microsomes Cytosolic enzymes are non-membrane-bound and occur free within the cytoplasm. They are generally associated with Phase II reactions, although some oxidation and reduction enzymes are contained in the cytosol. The most important enzyme system involved in Phase I reactions is the cytochrome P-450 enzyme system "mixed function oxidase (MFO) " system. It is found in microsomes and is responsible for oxidation reactions of a wide array of chemicals.

Modifiers of Biotransformation
The relative effectiveness of biotransformation depends on several factors, including species, age, gender, genetic variability, nutrition, disease, exposure to other chemicals that can inhibit or induce enzymes, and dose levels Differences are normally the basis for selective toxicity, used to develop chemicals effective as pesticides but relatively safe in humans Malathion in mammals is biotransformed by hydrolysis to relatively safe metabolites, but in insects, it is oxidized to malaoxon, which is lethal to insects.

Contd
Age may affect the efficiency of biotransformation Human fetuses and neonates (newborns) have limited abilities for xenobiotic biotransformations inherent deficiencies in many, but not all, of the enzymes responsible for catalyzing Phase I and Phase II biotransformations Biotransformation capability is also decreased in the aged Gender may influence the efficiency of biotransformation for specific xenobiotics limited to hormone-related differences in the oxidizing cytochrome P-450 enzymes.

Contd
Genetic variability in biotransforming capability accounts for most of the large variation among humans The Phase II acetylation reaction in particular is influenced by genetic differences in humans The most serious drug-related toxicity occurs in the slow acetylators "slow metabolizers acetylation is so slow that blood or tissue levels of certain drugs (or Phase I metabolites) exceeds their toxic threshold

Contd
Poor nutrition can have a detrimental effect on biotransforming ability inadequate levels of protein, vitamins, and essential metals decrease the ability to synthesize biotransforming enzymes Enzyme inhibition exposure to a substance will inhibit the biotransformation capacity for another chemical due to inhibition of specific enzymes Enzyme induction enhanced capability for biotransforming a xenobiotic increase the production of some enzymes increased level of enzyme activity increased biotransformation of a chemical subsequently absorbed. The most commonly induced enzyme reactions involve the cytochrome P-450 enzymes

Contd
Dose level can affect the nature of the biotransformation the biotransformation may be quite different at high doses versus that seen at low dose levels can be explained by the existence of different biotransformation pathways. At low doses, a xenobiotic may follow a biotransformation pathway that detoxifies the substance If the amount of xenobiotic exceeds the specific enzyme capacity, the biotransformation pathway is "saturated

Contd
a dose-related difference in biotransformation occurs with acetaminophen At normal doses, approximately 96% of acetaminophen is biotransformed to non-toxic metabolites by sulfate and glucuronide conjugation At the normal dose, about 4% of the acetaminophen is oxidized to a toxic metabolite toxic metabolite is conjugated with glutathione and excreted With 7-10 times the recommended therapeutic level, the sulphate and glucuronide conjugation pathways become saturated and more of the toxic metabolite is formed the glutathione in the liver may also be depleted so that the toxic metabolite is not detoxified and eliminated react with liver proteins and cause fatal liver damage.