PHM 450 exam 1

Categories of Toxicity

A fairly arbitrary system of toxicity ranking has evolved based on the LD50 of a substance. -A substance with an LD50 of less than 1 mg/kg is considered to be extremely toxic, while various definitions of highly toxic, moderately toxic and slightly toxic have been proposed. -Generally, highly toxic substances have an LD50 of less than 50 mg/kg, moderately toxic have an LD50 of less than 500 mg/kg and slightly toxic have an LD50 of greater than 500 mg/kg up to approximately 5 g/kg, which is the practical limit of most dosing techniques.

Age and sex

Age - Age may be important in determining the response to toxicants. Some chemicals are more toxic to infants or the elderly than to young adults. -For example: parathion is more toxic to young animals; nitrosamines are more carcinogenic to newborn or young animals. Sex - Although uncommon, toxic responses can vary depending on sex. - Examples are: male rats are 10 times more sensitive than females to liver damage from DDT; female rats are twice as sensitive to parathion as male rats

Assays to measure cytotoxicity

Assays that measure cytotoxicity fall into three general categories: 1) assays that measure the plasma membrane integrity 2) assays for mitochondrial function 3) assays that measure activation of pro-apoptotic enzymes

isochromosomes

Chromosomes with identical arms Form when centromeres divide along the incorrect plane during meiosis

Dermal

Dermal administration may be considered for substance that might be handled by workers or for cosmetics applied to the skin. -The test substance is painted onto the skin, covered with a patch of gauze held with tape and plastic is wrapped around the body to prevent ingestion of the substance.

Descriptive animal toxicity

Descriptive animal toxicity testing assumes that the effects produced by a compound in laboratory animals, when properly qualified, are applicable to humans, and that exposure o experimental animals to toxic agents in high doses is a necessary and valid method o discovering possible hazards in humans. -exposure o experimental animals to toxic agents in high doses is a necessary and valid method o discovering possible hazards in humans because the incidence o an e ect in a population is greater as the dose or exposure increases PAGE 16 OF BOOK

Oral

Dosing through the mouth is technically described as the peroral or per os (po) method. In some cases, the substance to be tested may be added directly to the animal's food or drinking water. -Alternatively, the test compound may be dissolved in water, vegetable oil (typically corn oil) or another vehicle (depending upon the solubility of the test substance) and introduced directly into the stomach through the use of a curved needle-like tube (this process is known as gavage). -Route of exposure of choice for substances that might be ingested in drinking water or food or taken orally as a drug.

Facilitated diffusion

Facilitated diffusion is similar to active transport because 1) there are specific transporters involved and 2) the process may be saturated or inhibited. However, unlike active transport, facilitated diffusion moves compounds down a concentration gradient, and does not require metabolic energy.

filtration

Filtration occurs when small molecules cross the cell membrane through pores down a concentration gradient. -By nature, molecules that are filtered must be: -small (MW ≤ 100) - water soluble (hydrophilic) - non-ionized -Pore sizes vary by cell and tissue type, with larger pores found in the kidney and sinusoidal membranes of the liver

Comparing toxicity of various substances

For some toxicants a small increase in dose causes a large increase in response (Toxicant A, steep slope). For other toxicants a much larger increase in dose is required to cause the same increase in response (Toxicant B, shallow slope).

Fractioning

Fractionating a total dose usually decreases the probability that the total dose will cause toxicity -sometimes the body can repair damage from each subtoxic dose if sufficient time passes before receiving the next dose -in such a case, the total dose, which would be harmful if received all at once, is non-toxic when administered over a period of time -For example, 30 mg of strychnine at one time could be fatal to an adult whereas 3 mg of strychnine each day for ten days would not

Hormesis

Hormesis, a "U-shaped" dose-response curve, results with some xenobiotics that impart beneficial or stimulatory effects at low doses but adverse effects at higher doses.

Live and dead kangaroo rat (PtK2) cells are evident in this picture, with live cells stained green and dead cells stained red. What is the likely combination of dyes used for this experiment?

In this assay, live cells are green and dead cells are red. They were stained with a combination of ethidium homodimer-1 (red) and calcein-AM (green)

mitochondrial DNA

Mitochondrial DNA contains 37 genes, all of which are essential for normal mitochondrial function. Thirteen of these genes provide instructions for making enzymes involved in oxidative phosphorylation. The remaining genes provide instructions for making molecules called transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs), which are chemical cousins of DNA. These types of RNA help assemble protein building blocks (amino acids) into functioning proteins

Orfila

Orfila (1787-1853): -a Spanish physician - the first to publish a comprehensive work (in 1815) devoted expressly to the toxicity of natural agents -demonstrated effects of poisons on specific organs by analyzing autopsy materials for poisons and their associated tissue damage. -Orfila was the first toxicologist to use autopsy material and chemical analysis as legal proof of poisoning and his work survives today as the basis of modern forensic toxicology

Paracelsus

Philippus Aureolus Theophrasutus Bombastus von Honhenheim-Paracelsus (1493-1541): - a physician-alchemist -determined that specific chemicals were actually responsible for the toxicity of a plant or animal poison - "toxicon" -documented that the body's response to those chemicals depended on the dose received -his studies revealed that small doses of a substance might be harmless or beneficial whereas larger doses could be toxic. This is now known as the dose-response relationship - a major concept of toxicology. -Paracelsus is often quoted for his statement: "All substances are poisons; there is none which is not a poison. The right dose differentiates a poison from a remedy."

First pass effect

Presystemic Elimination (First Pass Effect)

Quantitative toxicology

Quantitative toxicology involves challenging test animals with the substance being evaluated, which is applied in an ordered series of doses -The dose is controlled by the toxicologist; therefore, it is considered to be the independent variable -Response of the animals may be measured in many different ways, and is generally dependent upon the dose administered (i.e. it is the dependent variable)

Xenobiotic storage

Storage of toxicants in body tissues sometimes occurs. -Initially, when a toxicant enters the blood plasma, it may be bound to plasma proteins. This is a form of storage since the toxicant, while bound to the protein, does not contribute to the chemical's toxic potential. -Albumin is the most abundant and important plasma protein that binds toxicants. Normally, the toxicant is only bound to the albumin for a relatively short time. -The primary sites for toxicant storage are adipose tissue, bone, liver and kidneys.

Cellular response to injury tertiary events

Tertiary events are the final observable events in cellular toxicity, and include: -Steatosis - Membrane blebbing -Apoptosis - Necrosis - Tertiary events may or may not include cell death via apoptosis or necrosis, and may or may not be reversible, depending on the extent of damage.

Modern Toxicology

The 21st century is marked by an advanced level of understanding of toxicology -The discovery of DNA and various biochemicals that maintain body functions allowed us to study toxic effects on organs and cells at the molecular level -It is recognized that virtually all toxic effects are caused by changes in specific cellular molecules and biochemicals

Passage across cell membranes

The ability of a toxicant to cross the cell membrane is a key step in cellular toxicity, since many toxicants must enter the cell to trigger toxicity. There are five primary mechanisms by which a compound can cross a cell membrane. The five mechanisms are: - Filtration - Passive diffusion -Active transport - Facilitated diffusion -Endocytosis

gram

The gram is the standard unit -However, most exposures will be smaller quantities and thus the milligram (mg) is commonly used. For example, the common adult dose of ibuprofen (Advil) is 400 mg -The clinical and toxic effects of a dose must be related to body size -For example, 400 mg is the adult dose of ibuprofen; a dose that would be quite toxic to children (who are much smaller) -Therefore, the most commonly used dose metric is the amount of a substance administered on a body weight basis (also known as the dosage) -most common dosage measurement is mg/kg which stands for mg of substance per kg of body weight

p53

The suppressor gene most frequently altered in human tumors is the p53 gene. Damaged p53 genes have been identified in over 50% of human cancers. The p53 gene normally halts cell division and stimulates repair enzymes to rebuild and restore the damaged regions of the DNA. If the damage is too extensive, the p53 commands the cell to selfdestruct. An altered p53 is incapable of these defensive actions and can not prevent the cell with damaged DNA from dividing and proliferating in an erratic and uncontrolled manner. This is the essence of cancer. Recent studies have shown that an altered p53 can be inherited and affected new borne babies are thus susceptible (at higher risk) for certain types of cancer for the remainder of their lifetime.

dose vs dosage

The terms dose and dosage are often used interchangeably -dose commonly refers to the amount of substance administered -dosage refers to the amount of a substance administered per unit body weight of the test subject. -Therefore, the dose of a drug might be expressed in mg, while the dosage would be expressed as mg/kg of body weight. -In toxicity testing, compounds are administered as dosages, which allows better standardization of the amount of chemical administered. -In respiratory exposures, exposure levels are usually measured by the concentration of the substance in the environment (mg/m3 ).

factors influencing toxicity

The toxicity of a substance depends on: - form and innate chemical activity - dosage, especially the dose-time relationship - exposure route -species -age - sex -disposition (often referred to as toxicokinetics) -absorption (ability to be absorbed) -distribution (within the body) - metabolism -excretion - presence of other chemicals

Margin of Safety

The use of the ED50 and LD50 doses to derive the TI may be misleading as to safety, depending on the slope of the dose response curves for therapeutic and lethal effects. -To overcome this deficiency, toxicologists often use another term to denote the safety of a drug - the Margin of Safety (MOS). -The MOS is usually calculated as the ratio of the dose that is just within the lethal range (LD01) to the dose that is 99% effective (ED99). MOS = LD01/ED99 -A physician must use caution in prescribing a drug in which the MOS is less than 1. -you may also see the toxic dose (TD01) used instead of the lethal dose.

Types of target organ toxicity

Types of organ specific toxic effects are: - Blood/Cardiovascular toxicity - Dermal/Ocular toxicity -Genetic toxicity (germ cells) -Hepatotoxicity - Immunotoxicity - Nephrotoxicity -Neurotoxicity -Reproductive toxicity -Respiratory toxicity

Understanding volume of distribution

Vd = total amount of toxicant in body/concentration of toxicant in blood plasma -Vd is a theoretical value that tells you where the toxicant is distributed in body -Total body water has three compartments: plasma water, interstitial water, intracellular water (extracellular water = plasma water + interstitial water) -If Vd = plasma volume, then toxicant is likely in blood only -If Vd < than plasma volume, it has spread beyond the plasma, and/or may be stored in storage depots

chemical interactions

Xenobiotics administered or received simultaneously may act independently of each other. However, in many cases, the presence of one chemical may drastically affect the response to another chemical. -The toxicity of a combination of chemicals may be less or it may be more than would be predicted from the known effects of each individual chemical. -The effect that one chemical has on the toxic effect of another chemical is known as an interaction. -Chemical interactions described can be categorized by their chemical or biological mechanisms as follows: -chemical reactions between chemicals -modifications in absorption, metabolism, or excretion -reactions at binding sites and receptors -physiological changes

Ammonia (pH 11) would be expected to be concentrated and excreted in breast milk. A) True B) False

a

Calcein and trypan blue are used in viability assays as a measure of: A) Cell membrane integrity B) DNA damage C) Cell proliferation D) Mitochondrial function

a

Given the choice between Chemical A and Chemical B - which is more potent? A) Chemical A B) Chemical B C) Potency is equal

a

Lipid peroxidation occurs when there is disruption of phospholipid covalent bonds. Phospholipid chains that have a higher number of unsaturated bonds are: A) More likely to undergo lipid peroxidation than saturated bonds B) Less likely to undergo lipid peroxidation than saturated bonds C) Neither statement is accurate

a

The vast majority of proteins encoded by tumor suppressor genes act as: A) inhibitors of cell proliferation B) activators of apoptosis C) activators of cell proliferation D) inhibitors of gap junction communication E) DNA-repair enzymes

a

Which of the following will increase the absorption of a toxicant? A) increased lipid solubility B) increased water solubility C) increased particle size D) decreased pH of the GI tract E) all of the above

a

chemical antagonism (inactivation)

a chemical reaction between two compounds that produces a less toxic product -arsenic toxicity can be reduced by chelating the arsenic ions with dimercaprol

Xenobiotic

a foreign substance taken into the body -Xenobiotics may produce beneficial effects or they may be toxic -A xenobiotic in small amounts may be non-toxic and even beneficial (e.g. pharmaceuticals) but when the dose is increased, toxic and lethal effects may result

tumor

a general term for the uncontrolled growth of cells that becomes progressively larger with time; tumors may be benign or malignant *also neoplasm

cancer

a malignant tumor that has the ability to metastasize or invade into surrounding tissue

Clinical toxicology

a medical science that is concerned with disease or illness caused by exposure to toxic substances -Clinical toxicologists are typically physicians that have undergone specialized training in areas including emergency medicine and poison management

Toxicant

a substance that produces an adverse biological effect of any nature (may be chemical or physical); effects can be of any type (i.e. acute, intermediate or chronic)

Toxin

a toxic substance that is produced by a biological organism (most exhibit immediate effects)

benign tumor

a tumor that does not metastasize or invade surrounding tissue

malignant tumor

a tumor that has the ability to metastasize or invade surrounding tissue (same as tumor) Most malignant tumors fall into one of two categories, carcinomas or sarcomas. The major differences between carcinomas and sarcomas are: carcinoma; malignant tumor arising in the epithelium most common form of cancer usually spread via the lymphatic system sarcoma: maligant tumor arising in muscle or connective tissue usually spread via the bloodstream frequently metastacizes to the lungs

metastasis

ability to establish secondary tumor growth at a new location away from the original site

Toxic agent

anything that can produce an adverse biological effect (may be chemical, physical or biological in form)

Biotransformation always results in a chemical being less toxic than the original chemical. A) True B) False

b

Exposure to a toxicant that induces CYP450 can lead to a situation in a patient wherein larger doses of a particular pharmaceutical must be administered to achieve a therapeutic blood level (due to increased metabolism of the pharmaceutical). This "situation" is known as: A) induction B) tolerance C) inhibition D) knockout E) none of the above

b

For some toxicants, oral toxicity is actually increased by diluting the dose. This is probably due to: A) the lack of competitive inhibition of xenobiotic transporters due increased dosage volume B) more rapid stomach emptying induced by increased dosage volume - which leads to more rapid absorption in the duodenum C) increased water solubility of lipid soluble compounds due to increased dosage volume

b

In toxicology testing, the material in which the test compound is dissolved prior to administration to the test subject is known as the: A) control B) vehicle C) dissolvant D) delivery

b

The body uses apoptosis to remove damaged cells, and to selectively remove cells during normal development. In an adult human, which of the following tissue types would be expected to use apoptosis for tissue repair? A) Female germ cells B) Gastrointestinal epithelium C) Neurons D) Retinal ganglion cells E) Cardiac muscle cells

b

The luciferase assay measures: A) Cell membrane integrity B) Cellular ATP content C) DNA damage D) Glutathione levels

b

The process that involves the clonal expansion of initiated cells to produce a preneoplastic lesion: A) genotoxicity B) promotion C) progression D) metastasis E) fixation

b

The target organ is always the site of the highest concentration of the chemical. A) True B) False

b

The toxicity of a compound is directly due to the amount of the toxicant that is: A) bound to plasma proteins B) unbound to plasma proteins C) bound to tissue (such as liver or kidney) D) stored in a depot (such as fat or bone) E) metabolized

b

There are several acquired characteristics essential for the development of all malignant tumors. These include which of the following: A) scheduled apoptosis B) tissue invasion C) sensitivity to anti-growth signals D) limited angiogenesis E) all of the above

b

Which of the following is NOT a characteristic of the blood-brain barrier? A) the brain capillary endothelial cells are tightly joined B) the brain capillary endothelial cells are insensitive to lipid-soluble toxicants C) the brain capillary endothelial cells contain ATP-dependent transporters that transport some chemicals back into the blood D) the brain capillaries are surrounded by astrocytes E) the protein concentration in the interstitial fluid is much lower than that of other body fluids

b

Which of the following would have the largest negative impact on intracellular ATP levels? A) Moderately decreased caloric intake B) Interference with electron delivery to the electron transport chain C) Inability to harvest ATP from glycolysis D) Increased synthesis of biomolecules active cell division

b

Which suspected human carcinogen is found in fried foods (such as french fries)? A) benzene B) acrylamide C) methylene chloride D) acrylonitrile E) dioxin

b

p53 is a tumor suppressor gene that functions by: A) repairing DNA breaks B) arresting the cell cycle C) inducing CYP450 D)Inhibiting CYP450

b

A chemical carcinogen that requires metabolic activation to become carcinogenic is known as a(n): A) initiator B) electrophile C) procarcinogen D) genotoxic carcinogen

c

A highly lipophilic toxicant would be expected to be stored in: A) The lungs B) The liver C) Adipose tissue D) Skeletal muscle

c

A toxic substance produced by a biological organism is generally known as a: A) poison B) toxicant C) toxin D) venom E) none of the above

c

An event that results in a carcinogen-induced mutation is known as: A) progression B) promotion C) initiation D) metastasis

c

At low doses, 2,3,7,8-tetrachlorodibenzo(p)dioxin (dioxin) has been postulated to inhibit certain types of estrogen-dependent cancers. This is an example of: A) potentiation B) dispositional antagonism C) hormesis D) chronic toxicity E) none of the above

c

Carbon tetrachloride causes secondary damage to: A) Mitochondria B) Cell membrane C) Endoplasmic reticulum D) Cytoskeleton

c

Given the choice between Chemical A and Chemical B - which has higher maximal efficacy? A) Chemical A B) Chemical B C) Efficacy is equal

c

Mitochondria are critical targets of toxicants primarily because: A) Mitochondria control cellular protein synthesis B) Mitochondria contain critical transporters for carrying molecules across the plasma membrane C) Mitochondria produce cellular ATP needed to perform basic cellular functions D) Mitochondria are more prone to damage from lipid peroxidation than other organelles

c

The MTT assay measures: A) Cell membrane integrity B) DNA damage C) Mitochondrial function D) Glutathione levels

c

What is the LD50 for chemical X, based on the figure below? A) 7 mg B) 12 mg C) 17 mg D) 20 mg E) 25 mg

c

Which organ is most important when considering biotransformation of a chemical? A) Skin B) Lungs C) Liver D) Kidneys

c

dicentric chromosomes

chromosomes with 2 centromeres due to a chromosomal rearrangement

Mechanistic toxicologists

concerned with identifying and understanding cellular, biochemical and molecular mechanisms of toxicants

A sustained elevation of intracellular ____________ is usually an initiating step in cell death. A) Magnesium B) Potassium C) Sodium D) Calcium

d

Knowledge of the dose-response relationship permits one to determine: A) the rate of excretion of the xenobiotic B) the degree of metabolism of a xenobiotic C) the relationship of exposure dose to absorbed dose D) the threshold for the effect E) all of the above

d

The activation of a proto-oncogene into an oncogene involves which of the following: A) translation B) metabolism C) progression D) mutation

d

The primary method by which toxicants are identified as potential carcinogens: A) mouse skin model B) mouse lymphoma assay C) Ames assay D) 2-year rodent bioassay

d

Cytochrome c is an important molecule in initiating apoptosis in cells. All of the following regarding cytochrome c are true EXCEPT: A) The release of cytochrome c into the cytoplasm is an important step in apoptosis initiation. B) The loss of cytochrome c from the electron transport chain blocks ATP synthesis by oxidative phosphorylation. C) Loss of cytochrome c from the inner mitochondrial membrane results in increased formation of reactive oxygen species. D) Bax proteins mediate cytochrome c release. E) Caspases are proteases that increase cytoplasmic levels of cytochrome c.

e

Which of the following can increase the absorption of toxicants through the skin? A) increased stratum corneum hydration B) damaged stratum corneum C) increased skin temperature D) small size of the toxicant E) all of the above F) none of the above

e

Which of the following is NOT an outcome of acute toxicity testing: A) identification of target organs B) identification of the LD50 C) identification of species differences D) provide guidance for the selection of doses for subsequent testing E) provide guidance for selection of the route of exposure for subsequent testing

e

Environmental Toxicology

focuses on the impacts of chemical pollutants on biological organisms (including humans) -Ecotoxicology is a specialized area of environmental toxicology that focuses more specifically on the impacts of environmental pollutants on ecosystems

The first pass effect is important for toxicants administered through which route(s) of administration? A) oral B) inhalation C) intraperitoneal D) intravenous E) all of the above F) A & B only G) A & C only H) A & D only I) A, B & C only

g

Once initiated cells are produced, the following outcomes are possible: A) the initiated cell can remain in a non-dividing state B) the initiated cell may undergo apoptosis C) the initiated cell may undergo cell division (leading to proliferation) D) the initiated cell may undergo DNA repair and revert to a normal state E) A and B only F) A and C only G) B and C only H) A, B and C only I) A and D only J) all of the above

h

Forensic toxicology

hybrid of analytic chemistry and fundamental toxicological principles -concerned primarily with medical and legal aspects of toxicology

toxic dose

indicate doses that cause adverse toxic effects (TD) -TD0: toxic to 0% of the population -TD10: toxic to 10% of the population -TD50: toxic to 50% of the population -TD90: toxic to 90% of the population

stages of cancer development

initiation, promotion, progression

NOAEL

no observed adverse effect level -the highest data point at which there was not an observed toxic or adverse effect

nonmonotonic

non-monotonic curves, in contrast, change direction. Over part of the curve, response increases with dose, while over another portion it decreases as dose increases. Non-monotonic curves are often called 'inverted-U' (upper) or 'U'(lower).

Descriptive toxicologists

primarily involved in toxicity testing

Regulatory toxicologists

responsible for deciding, on the basis of the available data generated by descriptive and mechanistic toxicologists, whether a drug or other chemical poses a sufficiently low risk to be marketed for a stated purpose

toxicologist

scientist who is trained to examine and communicate the nature of xenobiotic induced effects on human, animal and environmental health

toxic substance

simply a material which has toxic properties -may be a discrete toxic chemical or a mixture of toxic chemicals -Hexane is a discrete toxic chemical -Asbestos is a toxic material that does not consist of an exact chemical composition but a variety of fibers and minerals -Gasoline is also a toxic substance but is distinct from a toxic chemical in that it contains a mixture of many chemicals -Toxic substances may not always have a constant composition. For example, the composition of gasoline varies with octane level, manufacturer, season, etc. -cigarette smoke

absorbed dose

the actual amount of the exposed or administered dose that is absorbed by the body

exposure dose

the amount of a xenobiotic encountered in the environment

tissue dose

the amount of absorbed dose that reaches a particular target organ or tissue

excretion

the elimination of a substance (or metabolites) from the body -The site and rate of excretion is another major factor affecting the toxicity of a xenobiotic. -The kidney is the primary excretory organ, followed by the gastrointestinal tract, and the lungs (for gases). -Xenobiotics may also be excreted in sweat, tears, and milk. -A large volume of blood serum is filtered through the kidney. -Lipid-soluble toxicants are reabsorbed and concentrated in kidney cells. -impaired kidney function causes slower elimination of toxicants and increases their toxic potential.

neoplasia

the growth of new tissue with abnormal and unregulated cellular proliferation

LOAEL

the lowest data point at which there was an observed toxic or adverse effect

Distribution

the movement of a substance from the site of absorption to other sites within the body -The distribution of toxicants and toxic metabolites throughout the body ultimately determines the sites where toxicity occurs. -A major determinant of how a toxicant will distribute, and whether or not a toxicant will damage cells is its lipid solubility. -If a toxicant is lipid-soluble it readily penetrates cell membranes. -Many toxicants are stored in the body. -Fat tissue, liver, kidney, and bone are the most common storage depots. - Blood serves as the main avenue for distribution. Lymph also distributes some materials.

Absorption

the movement of a substance into the bloodstream -The ability to be absorbed is essential for systemic toxicity to occur. Some chemicals are readily absorbed and others poorly absorbed. -Nearly all alcohols are readily absorbed when ingested, whereas there is virtually no absorption for most polymers -The rates and extent of absorption may vary greatly depending on the form of the chemical and the route of exposure -Ethanol is readily absorbed from the gastrointestinal tract but poorly absorbed through the skin.

carcinogenesis

the production of a carcinoma (epithelial cell cancer); general term used to describe the production of any type of tumor

administered dose

the quantity administered (usually orally or via injection)

reference dose

the reference dose is calculated from the NOAEL by adding a series of safety factors. These safety factors take into account uncertainties in extrapolating animal research to human, as well as differences insensitivity among groups of people, and between kids and adults. Thus if the NOAEL is found to be 1 milligram per kilogram of bodyweight per day (which corresponds to apart per million), then the reference dose might be 1 part per billion per day

Disposition/Toxicokinetics

the study of "how a substance gets into the body and what happens to it in the body" -the quantitative characterization of xenobiotic distribution in the body -Absorption -Distribution -Metabolism -Excretion

Toxicology

the study of the adverse effects of xenobiotics on living organisms -These adverse effects may occur in many forms -ranging from immediate death to subtle changes not realized until months or years later -They may occur at various levels within the body -an organ, a cell or a specific molecule or protein

total dose

the sum of all individual doses

metabolism

the transformation of a substance into another substance (metabolites) -Metabolism, also known as biotransformation, is a major factor in determining toxicity. -The products of metabolism are known as metabolites. -There are two types of metabolism: detoxification and bioactivation. -Detoxification is the process by which a xenobiotic is converted to a less toxic form. This is a natural defense mechanism of the organism. Generally the detoxification process converts lipid-soluble compounds to polar compounds. -Bioactivation is the process by which a xenobiotic may be converted to more reactive or toxic forms.

Poison:

toxicant or toxin that produces immediate death or illness when encountered in very small amounts

The figure to the right shows cumulus cells that have been frozen & thawed, then incubated in trypan blue and viewed under a microscope (x320). The trypan blue assay is often called an "exclusion assay." If the dye is excluded from the cell, is the cell alive or dead?

trypan blue is excluded from cells with intact cell membranes. If the membrane is compromised, the cell is dead, and the dye may enter the cell. -Arrows in Figure A show dead cells that have taken up trypan blue inside the cell.

effective dose

used to indicate the effectiveness of a substance (ED) -Normally, effective dose refers to a beneficial effect (e.g. relief of pain). It might also stand for a harmful effect (e.g. CNS depression). Thus the specific endpoint must be indicated. -ED0: effective for 0% of the population -ED10: effective for 10% of the population -ED50: effective for 50% of the population -ED90: effective for 90% of the population

potentiation

when one substance does not have a toxic effect on a certain organ or system but when added to another chemical makes that chemical much more toxic. -Potentiation occurs when a chemical that does not have a specific toxic effect makes another chemical more toxic. -Examples are: -The hepatotoxicity of carbon tetrachloride is greatly enhanced by the presence of isopropanol. -Normally, warfarin (a widely used anticoagulant in cardiac disease) is bound to plasma albumin so that only 2% of the warfarin is active. Drugs which compete for binding sites on albumin increase the level of free warfarin causing fatal hemorrhage.

ring chromosomes

when telomeres break, becoming sticky and the chromosome connects at each ends with each other. Can cause symptoms

synergism

when the combined effects of two chemicals are much greater than the sum of the effects of each agent given alone. -Synergism can have serious health effects. With synergism, exposure to a chemical may drastically increase the effect of another chemical. -Examples are: -Exposure to both cigarette smoke and radon results in a significantly greater risk for lung cancer than the sum of the risks of each. -The combination of exposure to asbestos and cigarette smoke results in a significantly greater risk for lung cancer than the sum of the risks of each. -The hepatotoxicity of a combination of ethanol and carbon tetrachloride is much greater than the sum of the hepatotoxicity of each.

dispositional antagonism

when the disposition (absorption, distribution, metabolism or excretion) of a chemical is altered so that the concentration and/or duration of the chemical at the target organ are diminished - altering the pH of urine will increase excretion of a variety of compounds

functional antagonism

when two chemicals counterbalance each other by producing opposite effects on the same physiologic function - a severe drop in blood pressure due to a barbituate overdose can be reversed by administration of a vasopressor to increase blood pressure

receptor antagonism

when two chemicals that bind to the same receptor produce less of an effect when given together than the addition of their separate effects of when one chemical antagonizes the effect of the second chemical -Naloxone blocks opioid receptors ... used in morphine/heroine overdose to combat CNS depression

mutations are recessive or dominant

A fundamental genetic difference between organisms is whether their cells carry a single set of chromosomes or two copies of each chromosome. The former are referred to as haploid; the latter, as diploid. Most simple unicellular organisms are haploid, whereas complex multicellular organisms (e.g., fruit flies, mice, humans) are diploid. Different forms of a gene (e.g., normal and mutant) are referred to as alleles. Since diploid organisms carry two copies of each gene, they may carry identical alleles, that is, be homozygous for a gene, or carry different alleles, that is, be heterozygous for a gene. A recessive mutation is one in which both alleles must be mutant in order for the mutant phenotype to be observed; that is, the individual must be homozygous for the mutant allele to show the mutant phenotype. In contrast, the phenotypic consequences of a dominant mutation are observed in a heterozygous individual carrying one mutant and one normal allele. Recessive mutations inactivate the affected gene and lead to a loss of function. For instance, recessive mutations may remove part of or all the gene from the chromosome, disrupt expression of the gene, or alter the structure of the encoded protein, thereby altering its function. Conversely, dominant mutations often lead to a gain of function. For example, dominant mutations may increase the activity of a given gene product, confer a new activity on the gene product, or lead to its inappropriate spatial and temporal expression. Dominant mutations, however, may also be associated with a loss of function. In some cases, two copies of a gene are required for normal function, so that removing a single copy leads to mutant phenotype. Such genes are referred to as haplo-insufficient. In other cases, mutations in one allele may lead to a structural change in the protein that interferes with the function of the wild-type protein encoded by the other allele. These are referred to as dominant negative mutations.

Gene

A gene is the basic physical and functional unit of heredity. -Genes, which are made up of DNA, act as instructions to make molecules called proteins. - Alleles are forms of the same gene with small differences in their sequence of DNA bases.

Where is DNA found

Although most DNA is packaged in chromosomes within the nucleus, mitochondria also have a small amount of their own DNA.

chemical carcinogens

Although there is continued debate of the molecular mechanisms involved in carcinogenesis, there is no debate over the fact that certain chemicals can act as carcinogens. Evidence for this comes from both epidemiological and experimental animal studies. Carcinogens that have the ability to bind to and alter the structure of DNA are generally called genotoxic (or genetic) carcinogens, while carcinogens that bind to and affect other cellular targets are known as epigenetic carcinogens. Some compounds appear to be intrinsically carcinogenic, whereas others must undergo bioactivation to produce reactive metabolites. Although the many substances identified as chemical carcinogens have a very broad range of structures with no obvious unifying features, they can be classified into two broad categories: directacting and indirect-acting. Direct-acting carcinogens are highly electrophilic compounds that can react with DNA. Indirect-acting carcinogens must be metabolized before they can react with DNA. All these chemicals can act as mutagens.

genotype vs phenotype

Biologists (and toxicologists) often distinguish between the genotype and phenotype of an organism. Strictly speaking, the entire set of genes carried by an organism is its genotype, whereas the function and physical appearance of an organism is referred to as its phenotype. Genotype usually denotes whether an individual organism carries mutations in a single gene (or a small number of genes), and phenotype denotes the physical and functional consequences of that genotype.

genes and cancer

Cellular DNA contains two types of genes - structural genes and regulatory genes. Structural genes direct the production of specific proteins within the cell. Regulatory genes control the activity of the structural genes and direct the proliferation process of the cell. The three classes of regulatory genes considered to have major roles in the carcinogenesis process are known as: proto-oncogenes oncogenes tumor suppressor genes

complete carcinogens

Certain chemicals (called complete carcinogens) seem to be able to produce malignant tumors without administration of a second chemical. Other carcinogens appear to be only initiators and seem to require subsequent exposure to and action of a promoter in order to produce cancer. Conversely, many promoters seem to be inactive unless there was prior exposure to an initiator; however, some promoters have induced cancer when used at high enough doses without an initiator.

Direct-acting carcinogen

Direct-acting carcinogens, of which there are only a few, are reactive electrophiles (compounds that seek out and react with negatively charged centers in other compounds). By chemically reacting with nitrogen and oxygen atoms in DNA, these compounds modify certain nucleotides so as to distort the normal pattern of base pairing. If these modified nucleotides were not repaired, they would allow an incorrect nucleotide to be incorporated during replication.

Gene regulation

Each cell expresses, or turns on, only a fraction of its genes. The rest of the genes are repressed, or turned off. The process of turning genes on and off is known as gene regulation. Gene regulation is an important part of normal development. Genes are turned on and off in different patterns during development to make a brain cell look and act different from a liver cell or a muscle cell, for example. Gene regulation also allows cells to react quickly to changes in their environments. Although we know that the regulation of genes is critical for life, this complex process is not yet fully understood. Gene regulation can occur at any point during gene expression, but most commonly occurs at the level of transcription (when the information in a gene's DNA is transferred to mRNA). Signals from the environment or from other cells activate proteins called transcription factors. These proteins bind to regulatory regions of a gene and increase or decrease the level of transcription. By controlling the level of transcription, this process can determine the amount of protein product that is made by a gene at any given time.

epigenetic carcinogens

Epigenetic carcinogens: increase incidence of tumors, but without any mutagenic activity These compounds are generally considered to be acting in the promotion phase of carcinogenesis. One example of a compound that is likely to act through epigenetic mechanisms is TCDD (dioxin). TCDD displays highly specific affinity to a cytosolic receptor protein (the aryl hydrocarbon receptor, or AhR). Upon binding to AhR, the TCDD-AhR complex moves into the nucleus, binds to a DNA receptor binding site, and induces the transcription of several genes. This may lead to promotion of cell division through actions on various regulatory enzymes. TCDD may also act in synergy with carcinogens that require bioactivation as TCDD increases cytochrome P450 levels. Finally, TCDD may also promote the development of cancer through its action as an immunosuppressant.

mutations are not always bad

Evolution is based on mutations (heritable changes in DNA) Mutations may be silent, decrease function, or sometimes increase function

intro to mutagenesis

Genetic toxicology is the branch of toxicology that is concerned with the effects of chemical and physical agents on DNA and the genetic processes of living cells. Genetic toxicology broadly encompasses both genotoxicity and mutagenicity - and it is important to differentiate between the two. A mutation is a heritable change in the genotype of a cell and mutagenesis is a term used to describe those process leading to such heritable changes. Genotoxicity, on the other hand, is a term that covers a broad range of endpoints - much broader than mutagenicity. For example, genotoxicity includes such processes as unscheduled DNA synthesis, sister chromatid exchanges and DNA strand breaks. These are not mutagenic processes per se because they are not in themselves heritable (transmissible from cell to cell or generation to generation) but they result in damage to DNA which results in cellular alteration

hormones and cancer

Hormones play an important role in cancer and are thought to act through epigenetic mechanisms, such as influencing gene expression. Both endogenous and exogenous estrogens have been implicated in promotion of breast cancer, and therapeutic interventions that antagonize the actions of estrogen have been shown to reduce the risk of occurrence or reoccurrence of cancers. One drug that is commonly used to treat estrogen-dependent cancers is tamoxifen, which is a competitive inhibitor of the estrogen receptor

chromosomes

In humans, each cell normally contains 23 pairs of chromosomes, for a total of 46. Twenty-two of these pairs, called autosomes, look the same in both males and females. The 23rd pair, the sex chromosomes, differ between males and females. Females have two copies of the X chromosome, while males have one X and one Y chromosome. The 22 autosomes are numbered by size. The other two chromosomes, X and Y, are the sex chromosomes. This picture of the human chromosomes lined up in pairs is called a karyotype.

induced mutations

In order to increase the frequency of mutation in experimental organisms, researchers often treat them with high doses of chemical mutagens or expose them to ionizing radiation. Mutations arising in response to such treatments are referred to as induced mutations . Generally, chemical mutagens induce point mutations, whereas ionizing radiation gives rise to large chromosomal abnormalities.

Indirect-acting carcinogens

Indirect-acting carcinogens generally are unreactive, water-insoluble compounds. They can act as potent cancer inducers only after conversion to ultimate carcinogens by introduction of electrophilic centers. Such metabolic activation of carcinogens is carried out by enzymes that are normal body constituents. In animals, activation of indirect-acting carcinogens (bioactivation) often is carried out by liver enzymes that normally function to detoxify noxious chemicals (e.g., therapeutic drugs, insecticides, polycyclic hydrocarbons, and some natural products). Many of these compounds are so fat-soluble that they would accumulate continually in fat cells and lipid membranes and not be excreted from the body. The detoxification system works by converting such compounds to water-soluble derivatives, which the body can excrete

genotoxic carcinogens

Most genotoxic carcinogens directly interact with DNA. Most commonly, they induce mutations through their interactions with the purine and pyrimidine bases found in the DNA molecule. Alkylating agents are one of the best-characterized categories of chemical carcinogen. As discussed earlier, the term 'genotoxic' is often mistakenly used to mean 'mutagenic'. Not all genotoxic mechanisms are mutagenic.

how do mutations happen

Mutations arise spontaneously at low frequency owing to the chemical instability of purine and pyrimidine bases and to errors during DNA replication. Natural exposure of an organism to certain environmental factors, such as ultraviolet light and chemical carcinogens (e.g., aflatoxin B1), also can cause mutations. A common cause of spontaneous point mutations is the deamination of cytosine to uracil in the DNA double helix. Subsequent replication leads to a mutant daughter cell in which a T•A base pair replaces the wild-type C•G base pair. Another cause of spontaneous mutations is copying errors during DNA replication. Although replication generally is carried out with high fidelity, errors occasionally occur. The figure on the next slide illustrates how one type of copying error can produce a mutation. In the example shown, the mutant DNA contains nine additional base pairs

types of mutations

Mutations can involve large or small alterations in DNA. A mutation involving a change in a single base pair (often called a point mutation) or a deletion of a few base pairs generally affects the function of a single gene. Changes in a single base pair may produce one of three types of mutation: missense mutation nonsense mutation frameshift mutation

oncogenes

Oncogenes are altered or misdirected proto-oncogenes which now have the ability to direct the production of proteins within the cell that can change or transform the normal cell into a neoplastic cell. Most oncogenes differ from their proto-oncogenes by a single point mutation located at a specific codon (group of three DNA bases that encodes for a specific amino acid) of a chromosome. The altered DNA in the oncogene results in the production of an abnormal protein that can alter cell growth and differentiation. It appears that a single activated oncogene is not sufficient for the growth and progression of a cell and its offspring to form a cancerous growth. However, it is a major step in the carcinogenesis process

other carcinogens

Other potential epigenetic carcinogens include promoters of cell division, such as the phorbol esters and heavy metals. The mechanism by which metals act as carcinogens is unclear. Some evidence indicates that they may in fact interact with DNA (a genotoxic mechanism), but other evidence suggests an inhibition of DNA repair mechanisms. Cadmium, for example, is nonmutagenic in mutagenicity assays, but has been found to produce overexpression of a number of genes potentially involved in carcinogenesis and to interfere with DNA repair. Epigenetic mechanisms may also be responsible for the gain or loss of entire chromosomes, thus producing aneuploidy. Unlike genotoxic carcinogens, epigenetic toxicants causing aneuploidy do not act directly on DNA, but instead act on other cellular components involved in cell division (such as spindle fibers, for example). Other possible epigenetic mechansims include interactions of carcinogens either with promoters or with products of the thousands of other genes that are transcribed into RNA.

progression

Progression is the third recognized step and is associated with the development of the initiated cell into a biologically malignant cell population. In this stage, a portion of the benign tumor cells may be converted into malignant forms so that a true cancer has evolved. Individual cells in this final stage can break away and start new clones of growth distant from the original site of development of the tumor. This is known as metastasis. Carcinogenesis In the real world... While the three-stage pathogenesis scheme describes the basic sequence of events in the carcinogenesis process, the actual events that take place in these various steps are due to activities of specific genes within the DNA of the cells.

proteins

Proteins are large, complex molecules made up of hundreds or thousands of smaller units called amino acids, which are attached to one another in long chains.

Proto-oncogenes

Proto-oncogenes (or protoonocogenes) are normal or good cellular genes that encode and instruct the production of the regulatory proteins and growth factors within the cell or its membrane. The proteins encoded by proto-oncogenes are necessary for normal cellular cell growth and differentiation. Activation of a proto-oncogene can cause alteration in the normal growth and differentiation of cells, which leads to neoplasia. Several agents can activate proto-oncogenes. This is the result of point mutations or by DNA re-arrangements of the proto-oncogenes. The product of this proto-oncogene activation is an oncogene. Many proto-oncogenes have been identified and have usually been named after the source of their discovery, e.g., the K-ras proto-oncogene was named for Dr. Kirsten's discovery using a rat sarcoma virus. H-ras, c-myc, myb, and src are other examples of proto-oncogenes. The proto-oncogenes are not specific for the original species but have been found in many other species, including humans. These proto-oncogenes are present in many cells but remain dormant until activated. Either a point mutation or chromosomal damage of various types can induce activation. Once activated they become an oncogene.

mutations

The development and function of any organism is predominantly controlled by genes. Mutations can lead to changes in the structure or function of an encoded protein or to a decrease or complete loss of its expression. Because a change in the DNA sequence affects all copies of the encoded protein, mutations can be particularly damaging to a cell or organism. In contrast, any alterations in the sequences of RNA or protein molecules that occur during their synthesis are less serious because many copies of each RNA and protein are synthesized. Gene mutations are considered to be DNA sequence changes that are limited to a single gene; larger genomic changes are generally referred to as "chromosomal alterations" because they affect more than one gene.

the flow of genetic information

The flow of information from DNA to RNA to proteins is one of the fundamental principles of molecular biology. It is so important that it is sometimes called the "central dogma."

initiation

The initiation phase consists of the alteration of the DNA (mutation) of a normal cell, which is an irreversible change. The initiated cell has developed a capacity for individual growth. At this time, the initiated cell is indistinguishable from other similar cells in the tissue. The initiating event can consist of a single exposure to a carcinogenic agent or in some cases, it may be an inherited genetic defect. An example is retinoblastoma in which children are pre-disposed to develop the cancer and the defect is passed down through successive generations. The initiated cell (whether inherited or a newly mutated cell) may remain dormant for months to years and unless a promoting event occurs it may never develop into a clinical cancer case. It is suspected that most people have initiated cells that may never progress further

promotion/conversion

The promotion/conversion phase is the second major step in the carcinogenesis process in which specific agents (referred to as promoters) enhance the further development of the initiated cells. Promoters often, but not always, interact with the cell's DNA and influence the further expression of the mutated DNA so that the initiated cell proliferates and progresses further through the carcinogenesis process. The clone of proliferating cells in this stage takes a form consistent with a benign tumor. The mass of cells remains as a cohesive group and physically keeps in contact with each other

chromosomal alterations

The second major type of mutation involves large-scale changes in chromosome structure and can affect the functioning of numerous genes, resulting in major phenotypic consequences. Such chromosomal alterations (or abnormalities) can involve deletion or insertion of several contiguous genes, inversion of genes on a chromosome, or the exchange of large segments of DNA between nonhomologous chromosomes. Structural chromosomal changes can occur during the formation of egg or sperm cells, in early fetal development, or in any cell after birth. Pieces of DNA can be rearranged within one chromosome or transferred between two or more chromosomes. The effects of structural changes depend on their size and location, and whether any genetic material is gained or lost. Some changes cause serious medical problems, while others may have no effect on a person's health. It is important to note that many cancer cells have changes in their chromosome structure. These changes are not inherited; they occur in somatic cells during the formation or progression of a cancerous tumor.

where do carcinogens come from

There are both naturally occurring and synthetic carcinogens. Exposure to carcinogens can occur at work, in the diet or from other environmental exposures. Carcinogenesis can result from chronic exposure to low levels of certain carcinogens, and a long latent period may occur before clear manifestation of cancer. Cancer can also arise through exposure to ionizing radiation and certain cell-transforming viruses. Estimating potential carcinogenicity is a major component in the registration of drugs, food additives, pesticides and in the control of toxic substances used in research and manufacturing.

changes in chromosome structure include

Translocations Deletions Duplications Inversions Isochromosomes Dicentric chromosomes Ring chromosomes

tumor supressor genes

Tumor suppressor genes, sometimes referred to as antioncogenes, are present in normal cells and serve to counteract and change the proto-oncogenes and altered proteins that they are responsible for. The tumor suppressor genes serve to prevent a cell with damaged DNA from proliferating and evolving into an uncontrolled growth. They actively function to effectively oppose the action of an oncogene. If a tumor suppressor gene is inactivated (usually by a point mutation), its control over the oncogene and transformed cell may be lost. Thus the tumor-potential cell can now grow without restraint and is free of the normal cellular regulatory control.

carbon monoxide poisoning

- Carbon monoxide has 50 times greater affinity for hemoglobin than oxygen. - As a result any carbon monoxide present causes a reduction in the oxygen binding capacity with a left shift in the oxygenhemoglobin dissociation curve. -This decreases oxygen unloading in tissues.

Developmental toxicity

- Developmental toxicity pertains to adverse toxic effects to the developing embryo or fetus. This can result from toxicant exposure to either parent before conception or to the mother and her developing embryo-fetus. - Chemicals cause developmental toxicity by two methods: -they can act directly on cells of the embryo causing cell death or cell damage, leading to abnormal organ development; or - they can induce a mutation in a parent's germ cell which is transmitted to the fertilized ovum; some mutated fertilized ova develop into abnormal embryos.

In the figure to the right, investigators are using Fura-2 AM to measure the response of hippocampal neurons to a number of toxins. What ion are they measuring with this dye?

- Fura-2 AM is used to measure intracellular calcium concentrations in living cells -The AM group is cleaved once the dye is in the cell, trapping the dye inside the cell

Cellular response to injury secondary event

- Secondary events occur as a consequence of primary events, rather than being due to the direct action of the toxicant itself. -Secondary events include: - Changes in membrane structure and permeability - Changes in the cytoskeleton - Mitochondrial damage and ATP depletion -Changes in intracellular calcium regulation

changes in cytoskeleton

- When the cytoskeleton is compromised, the integrity of the cell's structure may become damaged. -The cytoskeletal architecture can be changed through directly toxic compounds, such as phalloidin, a component of the toxic mushroom A. phalloides (the "death cap" mushroom) that binds to actin filaments in muscle cells and interferes with muscle function. -The cytoskeleton can also be affected indirectly by compounds that disrupt intracellular calcium levels, since calcium is an important regulator of cytoskeletal integrity. -Endothelial cells under the microscope. Nuclei are stained blue with DAPI, microtubles are marked green by an antibody bound to FITC and actin filaments are labelled red with phalloidin bound to TRITC.

apoptosis affects biological processes

- morphogenesis - tissue homeostasis - eliminate damaged cells - eliminate cells infected by viruses - eliminate self-reactive clones from the immune system

Permeability Transition Pore (PTP)

-A channel spanning inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) - Key role during apoptosis -PTP gating responsible for osmotic swelling of the mitochondrial matrix compartment and OMM rupture - Cytochrome c released after rupture

overview of assays

-A number of assays are used to identify cells that are stressed, damaged and/or dead. -Such assays are usually called viability assays because they determine whether or not a cell is still viable (i.e., living and metabolically active). - Other cellular toxicity assays measure whether or not a cell has actually died as a result of toxic insult, and are called cytotoxicity assays. - Quite often, scientists use the term "viability assay" to describe assays that measure either the health of the cell or whether the cell is alive or dead, while the phrase "cytotoxicity assay" is reserved for assays that measure and possibly count the number of living and dead cells. -Depending on the laboratory, viability and cytotoxicity assays can be used to observe single cells in a culture dish, or may be automated using laboratory robotics to allow for high-throughput screening. - Such assays are typically performed in vitro, using either primary cells in culture or cell lines.

portal of entry effect

-A specialized type of target organ toxicity is known as the portal of entry effect. -A portal of entry effect is a local effect produced in the tissue or organ of first contact between a toxicant and the biological system. -Portal of entry effects are very common with inhaled reactive chemicals (such as chlorine) that cause irritation in the respiratory tract. -A toxicant with portal of entry effects may also be a systemic toxicant upon absorption from the entry site.

Antioxidants

-A third means of removing reactive oxygen species is the administration or endogenous formation of antioxidants that accept (remove) radicals from reactive species, thereby reducing or preventing lipid peroxidation. - Common antioxidants include vitamin E (α-tocopherol), vitamin K, cysteine and ascorbate. - Antioxidants can also provide at least partial protection against chemical toxicity that involves formation of free radicals; for example, in vivo administration of vitamin E provides partial, but not complete, protection against carbon tetrachloride-induced liver damage.

Why is ADME important?

-A toxicant can only exert toxic effects if it reaches its site of action -Concentration at site of action depends on ability of toxicant to enter the body, reach its site of action, and "stick around" long enough to have a toxic effect

Examples of systemic toxicity

-Acute toxicity -subchronic toxicity - Chronic toxicity -genetic toxicity -Carcinogenicity -Developmental toxicity

Volume of distribution

-After entering the blood, a toxicant may distribute throughout the body -Rate of distribution: determined primarily by blood flow rate and the rate of diffusion out of the capillary bed into the cells of an organ or tissue -Final distribution: depends largely on the affinity of a xenobiotic for various tissues -Volume of distribution (Vd): indicator of the volume in which the toxicant would need to be uniformly dissolved in order to produce the observed blood concentration -Affected by binding to plasma proteins, storage in adipose, etc.

Role of Mitochondria in Apoptosis

-Amplifies and mediates extrinsic apoptotic pathways - Plays important role in intrinsic pathways - Mitochondria involved in death signals due to: - oxidative stress - DNA damage - chemotherapeutic drugs

DNA properties

-An important property of DNA is that it can replicate, or make copies of itself. -Each strand of DNA in the double helix can serve as a pattern for duplicating the sequence of bases. - This is critical when cells divide because each new cell needs to have an exact copy of the DNA present in the old cell.

apoptosis

-Apoptosis is often referred to "programmed cell death" because it can occur naturally as part of the body's tissue maintenance, allowing for removal of damaged cells from tissues. - Apoptosis can also be triggered by toxic insults, which trigger the same cascade of proteinmediated cellular degradation that ultimately break down the cell's DNA and protein components. - Apoptosis has several defining characteristics: -Cells undergo shrinkage and chromatin condensation - Cytochrome c is released by mitochondria into the cytoplasm, where it activates caspases (the cellular proteins that mediate the cell death signal to the degradative enzymes) -Degradative enzymes are activates that break down the cell's DNA, protein and lipid content. The endonucleases that break down DNA do so in a regulated fashion that produces 180-base pair fragments that can be detected by gel electrophoresis. - Membrane blebbing is visible.

Distribution opposed by

-Binding to plasma proteins -Specialized barriers -Distribution to storage sites -Association with intracellular binding proteins -Export from cells

Biotransformtion

-Biotransformation is the metabolic conversion of endogenous and xenobiotic chemicals to more water soluble compounds -Changes compound properties from those favoring absorption (lipophilicity) to favoring excretion in urine or feces (hydrophilicity) -*Exception to this general rule: elimination of volatile compounds by exhalation -Often used interchangeably with metabolism

membrane blebbing

-Blebbing occurs when the integrity of the plasma membrane breaks down, resulting in uneven membrane structure. -Blebbing is reversible in its early stages, and often precedes any changes in plasma membrane permeability. - Severe cellular damage that causes significant blebbing can lead to plasma membrane rupture and cell death.

Xenobiotic storage in bone

-Bone is composed of proteins and the mineral salt hydroxyapatite -Bone contains a sparse blood supply but is a live organ. -During the normal processes that form bone, calcium and hydroxyl ions are incorporated into the hydroxyapatite-calcium matrix. -Several chemicals, primarily elements, follow the same kinetics as calcium and hydroxyl ions and therefore can be substituted for them in the bone matrix. -Strontium (Sr) or lead (Pb) may be substituted for calcium (Ca), and fluoride (F-) may be substituted for hydroxyl (OH-) ions. - Bone is continually being remodeled under normal conditions. -Calcium and other minerals are continually being resorbed and replaced, on the average about every 10 years. Thus, any toxicants stored in the matrix will eventually be released to reenter the circulatory system.

calcium dysregulation

-Brief, reversible calcium increases are a normal part of intracellular signaling; however, prolonged elevations of intracellular calcium can trigger apoptosis. -For this reason, scientists often measure intracellular calcium concentration ([Ca2+ ] i ) as a means of monitoring calcium-linked signaling pathways, as well as to measure loss of calcium regulation as an early indicator of cell death.

Carcinogenicity

-Carcinogenicity is a complex multistage process of abnormal cell growth and differentiation which can lead to cancer. - At least two stages are recognized: -Initiation: a normal cell undergoes irreversible changes -Promotion: initiated cells are stimulated to progress to cancer. -Chemicals can act as initiators or promoters. - The initial neoplastic transformation results from the mutation of the cellular genes that control normal cell functions. -The mutation may lead to abnormal cell growth. - It may involve loss of suppressor genes that usually restrict abnormal cell growth. -Many other factors are involved (e.g., growth factors, immune suppression, and hormones). - A tumor (neoplasm) is simply an uncontrolled growth of cells. -Benign tumors grow at the site of origin; do not invade adjacent tissues or metastasize; and generally are treatable. - Malignant tumors (cancer) invade adjacent tissues or migrate to distant sites (metastasis). They are more difficult to treat and often cause death.

Proteins That Regulate Apoptosis

-Caspases have critical role in apoptosis - Apoptosis defined as caspase-dependent cell death - Caspases = cysteine dependent aspartate specific proteases Referred to as caspase-n - n = order of caspase publication

Changes in intracellular calcium regulation

-Cells maintain tight control of intracellular calcium concentrations through active transport out of the cell as well as through sequestration inside mitochondria and the smooth endoplasmic reticulum. -A sustained rise in intracellular calcium often signals imminent cell death, since calcium causes a number of deleterious cellular effects, including: - activation of proteases that degrade cellular proteins - activation of phospholipases that degrade membrane phospholipids - activation of endonucleases that degrade DNA -alterations in the cytoskeleton - Intracellular calcium dysregulation is involved in the toxicity of a wide range of toxicants.

Protective mechanisms against cell injury

-Cells possess a number of mechanisms to defend themselves against toxic insult: -DNA repair - Glutathione - Superoxide dismutase -Antioxidants

ischemia

-Cells rely on a constant supply of oxygen to maintain active aerobic metabolism and cell viability. -Reduced oxygen supply is called hypoxia - causes significant cellular and tissue damage. - Ischemia is the cellular and tissue damage that occurs when blood supply is cut off, or the capacity of the blood to carry oxygen is reduced or abolished. -Ischemia can result from physical trauma that cuts off blood supply, or from a reduced capacity of the blood to carry oxygen, as occurs during carbon monoxide and nitrite poisoning. - Ironically, restoration of oxygen supply often causes more damage through reperfusion injury, in which the new oxygen supply produces highly damaging free radicals.

chronic toxicity

-Chronic toxicity represents cumulative damage to specific organ systems and takes many months or years to become a recognizable clinical disease. -Examples: -cirrhosis in alcoholics who have ingested ethanol for several years -chronic kidney disease in workers with several years exposure to lead -chronic bronchitis in long-term cigarette smokers - pulmonary fibrosis in coal miners (black lung disease)

Activation of pro-apoptosis enzymes - caspase-3

-Commercially available caspase activity kits measure the ability of activated caspases to cleave a substrate, producing a fluorescent product. -Such kits usually focus on caspase-3, since it is activated early in the initiation of apoptosis. -However, they are limited to measuring only apoptosis and not necrosis, since necrosis typically does not involve caspase activation.

Phase 2 reactions

-Conjugation involves addition of an endogenous moiety to a foreign molecule which may be a product of a Phase I reaction (but doesn't have to be). -Major Phase II reactions: conjugation with glucuronic acid, sulfate, glutathione or amino acids; acetylation; and methylation. -Enzymes involved are tranferases except in the case of amino acid conjugation where the first step is catalyzed by an acyl CoA synthetase (then a transferase is involved).

covalent binding

-Covalent binding occurs when a molecule binds irreversibly to another molecule. -Depending on the binding site, this can lead to irreversible changes in function, as with cyanide's binding to cytochrome C oxidase. -Irreversible binding to DNA can lead to mutagenesis and carcinogenesis in actively dividing cells. -However, covalent binding to non-functional sites on proteins may not affect protein function and thus may not be a toxic event.

DNA repair

-DNA damage occurs via a variety of mechanisms, including ultraviolet light and a number of chemical mutagens. -DNA damage may be lethal if it affects one or more critical proteins for cellular function. -If a cell is dividing, DNA mutations may contribute to loss of control over the cell division process, leading to cancer. Thus, it is critical for cells to have the capacity to repair DNA.

DNA

-DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms. -Nearly every cell in a person's body has the same DNA. - Most DNA is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria (where it is called mitochondrial DNA or mtDNA).

Quantal Dose-Response Curves

-Describes the distribution of responses to different doses in a population of individual organisms -Called "quantal" because the measurement is "does the individual have a specific response, yes or no" (it is quantitative, not qualitative)

Individual (Graded/Continuous) Dose-Response Curves

-Describes the response of an individual organism to varying doses of a chemical/toxic agent -Often called a "graded" response because the measured effect is graded (continuous) over a range of doses

enzyme inhibition

-Direct inhibition of enzymatic activity can lead to cellular damage of varying degrees of severity, depending on the importance of the enzyme. - Cyanide prevents mitochondrial respiration by inhibiting the cytochrome c oxidase (cytochrome aa3) enzyme of the mitochondrial electron transport chain, thereby preventing the cell from producing ATP through oxidative respiration. -Enzyme inhibition may be reversible or irreversible.

mitochondria response

-Disruption of: - mitochondrial inner transmembrane potential - permeability transition (PT) - Osmotic mitochondrial swelling -Rupture of the outer mitochondrial membrane - Release of proapoptotic proteins into cytoplasm: - Released proteins include cytochrome c - Activation of caspases and apoptosis-inducing factor (AIF) Loss of biochemical homeostasis

Standard Deviation

-Dose responses are commonly presented as mean +/- 1 S.D. (standard deviation), which incorporates 68% of the individuals -The variance may also be presented as two standard deviations, which incorporates 95% of the responses. -A large standard deviation indicates great variability of response. For example, a response of 15 +/- 8 mg indicates considerably more variability than 15 +/- 2 mg.

Relationship between effective dose & toxic dose response

-Due to differences in slopes and threshold doses, low doses may be effective without producing toxicity. Although more patients may benefit from higher doses, this is offset by the probability that toxicity or death will occur. -The relationship between the Effective Dose response and the Toxic Dose response is illustrated below:

Activation of pro-apoptosis enzymes - cytochrome C

-During the early stages of apoptosis, mitochondria release cytochrome C into the cytosol, where it then activates the caspase cascade that eventually degrades the cell from within. -To measure cytochrome C translocation to the cytosol, cells must be lysed, then fractionated to separate the cytoplasm from the mitochondria. - The location of cytochrome C is then measured using a cytochrome C-specific antibody via immunoblotting

endocytosis

-Endocytosis is the process by which the cell membrane forms a pocket around a molecule to draw it into the cell in a membrane-bound vesicle that then enters the intracellular lysosomal degradation system. - Energy-dependent process that is typically carried out by cells such as macrophages, and allows for the movement of particles and compounds into the cell without actually passing through the cell membrane. - Endocytosis carried out by phagocytic cells, such as macrophages, is called phagocytosis -Endocytosis is the mechanism by which asbestos enters the lungs. Once asbestos has been absorbed into the lung tissue, it has the potential to cause fibrosis and lung tumors

Active transport

-Energy-dependent process for moving molecules across the cell membrane. - By definition, active transport involves: - a specific membrane-bound carrier system (transporter) - a metabolic energy source to drive the process (most commonly ATP) - transport against a concentration gradient -Because active transport occurs using transporters, the process is 1) saturable, 2) allows for competition between transported molecules, and 3) may be inhibited by toxicants that decrease cellular energy production (if the transporter is ATP-dependent). -The chemical structure of the compound becomes critical in active transport, since its ability to mimic the chemical properties of the natural substrate(s) is critical to being recognized and moved by the transporter. - Lead (Pb2+) enters the body by active transport by using the calcium (Ca2+) transporters in the intestine -Herbicide paraquat has a similar structure to polyamines (such as putrescine and spermine) that are normally carried by transporters in Type I and Type II alveolar epithelial cells. When paraquat is present, the alveolar transporters actively move paraquat into lung tissue, where it is toxic.

general issues regarding metabolism

-Enzyme overlap - more than one enzyme can metabolize a compound -Substrate overlap - one enzyme has multiple substrates - Multiple metabolism pathways for individual compounds -Phase I and Phase II may occur more than once, and not necessarily as I, then II - Reaction rates vary, effecting metabolite concentration in tissues - Metabolism does not necessarily cause detoxification

Membrane permeability assays - exclusion assays

-Exclusion assays measure the ability of the intact plasma membrane of the cell to exclude dyes from inside the cell. When the cell membrane is damaged, such dyes cross the usually impermeable cell membrane, and are a good indicator of cell death. -The most widely used exclusion assay dye is trypan blue, which is normally excluded from the cell. - When the plasma membrane becomes compromised, trypan will enter the cell and the cell interior will become blue, a measure of cell death. -Trypan blue is widely used in laboratories to count cells during cell culture, and to rapidly assess viability of cell suspensions. -There are two important facts to keep in mind when using trypan blue: 1) some cell membranes are naturally "leaky", which means that a perfectly healthy, viable cell may become stained light blue by the dye; and 2) trypan is itself toxic to cells during long exposures (greater than 10 minutes for normal working concentrations)

exposure route

-Exposure route is important in determining toxicity. -Some chemicals may be highly toxic by one route but not by others. - Two major reasons are differences in absorption and distribution within the body. - Ingested chemicals, when absorbed from the intestine, distribute first to the liver and may be immediately detoxified (this is known as the first pass effect) -Inhaled toxicants immediately enter the general blood circulation and can distribute throughout the body prior to being detoxified by the liver. -Frequently there are different target organs for different routes of exposure.

Biotransformation is performed by enzymes

-Four broad categories of reactions: Expose or introduce a functional group, usually result in small increase in hydrophilicity: -Hydrolysis -Reduction -Oxidation Add a functional group, often large, that greatly increase hydrophilicity: -Conjugation

Absorption by the lungs

-Gases, vapors of volatile or volatilized liquids, and aerosols/particles -May pass through the nose -Gases may be retained by mucosa of nose if gas is very water soluble or reacts with surface components -If enter lungs, gases may reach alveolar space and diffuse into capillaries -Ability to enter the bloodstream based on the blood-to-gas partition coefficient -Blood carries gas throughout body -Particles enter lungs based on size (general rule: smaller particles travel farther) - *more detail to come when we discuss respiratory toxicology*

Genetic toxicity

-Genetic toxicity results from damage to DNA and altered genetic expression. This process is known as mutagenesis. -The genetic change is referred to as a mutation and the agent causing the change as a mutagen. If the mutation occurs in a germ cell the effect is heritable. There is no effect on the exposed person; rather the effect is passed on to future generations. - If the mutation occurs in a somatic cell, it can cause altered cell growth (e.g. cancer) or cell death in the exposed person.

Factors in apoptosis

-Genetically encoded process -Triggers inside or outside the cell: -Ligands - Defects in DNA repair system - Radiation or cytotoxic drug damage - Lack of survival signals - Viral infections - Developmental death signals - Triggers result in common death signals - Triggers cause same apoptosis process

Glutathione

-Glutathione is considered to be a cell's primary defense against oxidative damage. -Glutathione is a tripeptide molecule consisting of L-glutamyl-Lcysteinylgycine (GSH). - The presence of the cysteinyl group provides a highly nucleophilic thiol that easily interacts with electrophilic molecules, such as free radicals, peroxides and other oxidizing compounds. - GSH can bind to a reactive molecule to form a glutathione conjugate that is then excreted from the body. - Some glutathione conjugates are themselves toxic. For example, the now-banned agricultural fumigant ethylene dibromide (EDB) forms a glutathione conjugate that alkylates DNA, making it highly carcinogenic.

Typical Measurements

-In a typical study, body weight of the test animals is measured either daily or periodically. -Animals are observed for behavior and symptomology (such as tremors or convulsions, for example). -During the exposure period, animals are monitored closely for signs of toxicity, as well as the timing of the appearance of those symptoms. -A slow onset of symptoms, for example, may suggest that the substance is being metabolized to a more toxic metabolite. -Following the exposure period, the animals are sacrificed and necropsied. -Sections of tissue samples may be sliced on a microtome and examined under the microscope for evidence of histopathology (which is any abnormality in cell or tissue seen under a microscope). Tissue samples may also be analyzed for the presence of biochemical indicators of pathology.

Potential stages in the development of toxicity

-In order to be toxic, the toxicant must reach the site of exposure; react with a target molecule; and cause cellular dysfunction -Followed by repair or dysrepair

monotonic dose-response curve

-In standard toxicology, as the dose increases, so does the effect. Conversely as dose decreases, so does its impact. -never reverse direction

Inhalation

-In the laboratory, toxicologists use inhalation chambers to study the effects of airborne toxicants. -An inhalation chamber consist of one or more areas in which animals are held during exposure, along with some apparatus for delivery of the toxicant being tested. -static test systems: the toxicant is simply introduced and mixed into the atmosphere in a closed chamber. -dynamic test systems: air is constantly circulated through the exposure chamber, with the toxicant being introduced into the air as it enters the chamber. Gases may be directly mixed with incoming air; particles may be introduced either as a dry dust or suspended in droplets of water -The concentration of gases and the concentration and size of particles can be monitored by sampling within the chamber, and the level of exposure can be adjusted accordingly. -The chambers in which the animals are exposed may vary also. -whole body of the animal may be exposed to the toxicant - head or neck only -restraint of the animal may pose a problem, but the problems of disposition of the toxicant on the animal's coat and subsequent ingestion of the toxicant during grooming are avoided

Units

-In toxicology, time is an important aspect to consider. Especially important for exposures of several days or chronic exposures. Most common time unit is one day and thus, the usual dosage unit is mg/kg/day -Since some xenobiotics are toxic in much smaller quantities than the milligram, often smaller fractions of the gram are used, such as microgram (µg) -Environmental exposure units are expressed as the amount of a xenobiotic in a unit of the media. Mg/liter (mg/l) for liquids. Mg/gram (mg/g) for solids. Mg/cubic meter (mg/m3) for air -Smaller units are used as needed (e.g. µg/ml)

measuring mitochondria function

-Irreversible loss of mitochondrial function is a sign of cell death. - The two primary methods for determining mitochondrial functionality are: 1) measurement of the inner mitochondrial membrane potential, and 2) measurement of cellular ATP levels.

Kinetics terminoloigy

-Kinetics - describes the movement of chemicals -Toxicokinetics is the appropriate term for the study of the kinetics of all toxic substances. -Frequently the terms toxicokinetics, pharmacokinetics, or disposition may be found in the literature to have the same meaning. -Disposition - often used in place of toxicokinetics to describe the time-course of movement of chemicals through the body. -The disposition of a toxicant along with its biological reactivity are the factors that determine the severity of toxicity that results when a xenobiotic enters the body

Lipid peroxidation

-Lipid peroxidation is the process by which free radicals interact with unsaturated lipids (such as those found in cell membranes), causing damage to the lipids by breaking them down into smaller fragments. - Lipid peroxidation is initiated when free radicals attack unsaturated bonds of fatty acids, removing hydrogen atoms from the fatty acid chains and converting them into free radicals themselves. - Lipid peroxidation can occur at either the plasma membrane or at organelle membranes. -Carbon tetrachloride (formerly used as a dry cleaning solvent) causes damage to the endoplasmic reticulum through lipid peroxidation (the actual damage is a secondary event caused by the primary event of the lipid peroxidation), contributing to the liver damage that is characteristic of carbon tetrachloride toxicity

Xenobiotic storage in adipose tissue

-Lipid-soluble toxicants are often stored in adipose tissues. -Adipose tissue is located in several areas of the body but mainly in subcutaneous tissue -Lipid-soluble toxicants can be deposited along with triglycerides in adipose tissues -The lipids are in a continual exchange with blood and thus the toxicant may be mobilized into the blood for further distribution and elimination, or redeposited in other adipose tissue cells

Membrane permeability assays

-Membrane permeability assays either measure the ability of the cell to exclude external dyes (exclusion assays), or retain a dye within the cell (retention assay)

Mitochondrial damage and ATP depletion

-Mitochondria are critical targets, since they provide the majority of the ATP that powers cellular activity. - Mitochondrial respiration depends on maintenance of the dual membrane structure of this organelle; thus, mitochondrial damage (especially swelling) disrupts mitochondrial function and prevents the formation of ATP. - Loss of cellular ATP results in lack of energy for a number of cellular processes, including protein synthesis, lipid synthesis and gluconeogenesis. - ATP depletion also deprives energy to the transporters that maintain Na+, K+ and Ca2+ gradients across the cell. - Inhibition of mitochondrial respiration leads to rapid cell death, as is the case with cyanide. - Similarly, MPP+, the reactive metabolite of the neurotoxic compound MPTP, inhibits mitochondrial respiration at Complex I, resulting in rapid cell death. -MPP+ inhibits mitochondrial respiration - selectively destroys substantia nigra cells - model for Parkinson's disease

Measuring mitochondrial membrane potential - MTT

-Mitochondrial function can be assessed using colorimetric assays that measure the ability of the mitochondria to convert tetrazolium reagents, such as MTT, into a purple formazan salt that can viewed through a light microscope. - The number of cells that are purple is directly proportional to the number of healthy, viable cells. -However, the assay has the disadvantages of being lengthy (formazan formation takes 1-4 hours to develop), and of making the cells unusable for further experiments after the assay.

Toxic effects

-Most observable cellular changes and cell death are due to specific biochemical changes within the cell or in the surrounding tissue. - However, there are a few situations where a toxic chemical or physical agent can cause cell damage without actually affecting a specific chemical in the cell or its membrane: -Physical agents such as heat and radiation may damage a cell by coagulating their contents. - Impaired nutrient supply (such as glucose and oxygen) may deprive the cell of essential materials needed for survival.

necrosis

-Necrosis is a form of cell death that is distinguishable from apoptosis in that it is not controlled and orderly like apoptosis. -Necrotic cells often undergo swelling of the plasma membrane, nucleus and mitochondria. -Similar to apoptosis, necrotic cells undergo severe loss of intracellular calcium regulation, and ultimate loss of plasma membrane integrity, where the cell membrane ultimately lyses. - Some scientists believe that apoptosis and necrosis are on two ends of the cell death continuum, with necrosis being the cellular response to more severe, immediate injury than apoptosis

Barriers to disposition

-Organs or tissues differ in the amount of a chemical that they receive or to which they are exposed -This is primarily due to two factors: the volume of blood flowing through a specific tissue and the presence of special "barriers" to slow down toxicant entrance.

Measuring mitochondrial membrane potential - Luciferase

-Other mitochondrial functional assays measure the intracellular concentration of ATP to determine whether the cell has enough energy to function. -Luciferase is commonly used to measure ATP levels using bioluminescence -When in the presence of ATP, luciferase catalyzes the oxidation of luciferin and, as a by-product, produces light (luciferase is the basis of firefly luminescence). - The luciferase assay is widely used because it is rapid, very sensitive, and can be used in a high-throughput screening environment; however, the assay is limited because it does require the cell to be lysed to measure intracellular ATP.

Types of phase 1 reactions

-Oxidation: Major oxidations are: aromatic, aliphatic, alicyclic, heterocyclic, N-oxidation, S-oxidation, dealkylation. Other enzymes also catalyze Phase I reaction: microsomal flavin monooxygenases; amine oxidases, peroxidases and alcohol dehydrogenase -Reduction: Major reduction reactions are azo reduction and nitro reduction. The enzymes (reductases) are found in the flora of the GI tract as well as mammalian tissues. -Hydrolysis: Major hydrolysis reactions are ester and amide hydrolysis. These are catalyzed by a group of enzyems with overlapping substrate specificity and activity. Hydrazides can also undergo hydrolysis. -Hydration: Primarily for epoxides - which may undergo addition of water. -Dehalogenation: Cytochrome P450s can also catalyze reductive dehalogenation

Passive diffusion

-Passive diffusion is the most common mechanism by which foreign compounds cross the cell membrane. -Energy-independent process - Does not rely on cellular ATP -Influenced primarily by the chemical characteristics of the compounds themselves -3 key criteria: -there must be a concentration gradient across the membrane - the compound must be lipid soluble -the compound must be non-ionized -the degree of ionization of a compound will vary by the pH of the solution it is in -A weak acid is, by definition, primarily non-ionized, and thus will be easily absorbed in an acidic environment (such as the stomach), so long as it is also lipid-soluble. - Weak bases are primarily non-ionized in more alkaline environments, and will be absorbed more readily in such an environment Henderson-Hasselbalch equation: pH = pKa + log[A-]/[HA] In this equation, pKa = dissociation constant for an acid in its ionized (A-) and non-ionized (HA) forms Most orally administered drugs are weak acids

Distribution facilitated by

-Porosity of the capillary endothelium -Specialized transport across the plasma membrane -Accumulation in cellular organelles -Reversible intracellular binding

Membrane permeability assays - retention assays

-Retention assays work in the opposite manner, in that a fluorescent dye is loaded into the cell, where it becomes trapped and stays within the cell so long as the plasma membrane is intact. -A common retention dye is calcein AM which, similar to fura-2 AM, easily crosses the cell membrane in the AM form. Once inside the cell, esterases cleave the AM group and the dye becomes trapped inside the cell, where it fluoresces green. If the plasma membrane is compromised, the dye rapidly leaks out of the cell. - Like exclusion assays, retention assays are widely used because they are quick and easy; however, as mentioned above, some cell membranes are naturally leaky, so retention dyes are not perfect for every cell type. - Because calcein is a fluorescent dye, it is often used in conjunction with a second fluorescent dye that more clearly labels the dead cells, in order to allow for a full count of both live and dead cells. - For example, one commercially available kit uses a combination of calcein AM (to label viable cells green) and ethidium homodimer (a red dye that combines with DNA only when the nuclear membrane has been compromised). - Retention assays can also be based on the ability of the cell to retain its own internal components inside the cell membrane, such as assays that measure release of intracellular enzymes. - One such assay measures the release of lactose dehydrogenase (LDH) from the cell into the cell culture supernatant. - When used in vivo, measurement of LDH in the bloodstream is a common method for measuring liver cell damage.

Absorption through the skin

-Skin is primary physical barrier to entry of toxicants -Highly impermeable - to enter via skin, must pass through multiple layers of skin to reach blood supply in dermal layer - The rate determining barrier to absorption is the stratum corneum -Skin absorption is favored by : -Abrading the skin (compromising the stratum corneum integrity) -Increasing hydration of the stratum corneum -Increased temperature, which increases dermal blood flow -Low solubility of toxicant in vehicle Small toxicant size

When you open the link to the Sigma-Aldrich web page for the Caspase 3 Assay Kit, the "customers also purchased" section lists Staurosporine as a commonly purchased reagent, which is used as a positive control in these experiments - what is the purpose of using Staurosporine in this assay?

-Staurosporine is a potent inducer of apoptosis, and is often used as a positive control in apoptosis assays -Caspase 3 activity in apoptotic Jurkat cells. Apoptosis was induced in Jurkat human cells with 1 μg/ml staurosporine for 0, 1, 2, and 3 hours. The cells were lysed in 1X lysis buffer at a ratio of 100 μl per 107 cells. The caspase 3 activity in the lysate was determined using the Caspase 3 Colorimetric Assay Kit (CASP-3-C). Ten μl of each lysate was tested both with and without caspase 3 inhibitor, in a total reaction volume of 100 μl using 96 well multiwell plates. The substrate (Ac-DEVD-pNA) concentration was 200 μM, and the inhibitor (Ac-DEVDCHO) concentration was 0.05 μM. The assay was performed at pH 7.4 at 37 °C for 42 minutes

Steatosis

-Steatosis is the accumulation of fat within cells. - Steatosis is a common toxic response, and can be reversible. -Steatosis is a common event in alcohol exposure, whereby the liver accumulates fat droplets intracellularly. -Steatosis is reversible following mild exposures, but with repeated exposure and/or large doses, steatosis may become irreversible, leading to the fatty liver observed in many alcoholics. -Steatosis is most often observed in liver cells, as this organ plays a pivotal role in lipid metabolism. -Interestingly, steatosis can result from interference with lipid metabolism at a number of steps, including: - Increased lipid production (ethanol, hydrazine) - Decreased lipid metabolism (vitamin deficiency) -Decreased lipid excretion from the cell (tetracycline, puromycin)

Barriers to disposition - Blood-Brain Barrier

-Structural barriers exist that restrict entrance of toxicants into certain organs or tissues. The primary barriers are those of the brain, placenta, and testes. -The blood-brain barrier protects the brain from most toxicants. Specialized cells called astrocytes possess many small branches, which form a barrier between the capillary endothelium and the neurons of the brain. Lipids in the astrocyte cell walls and very tight junctions between adjacent endothelial cells limit the passage of water-soluble molecules. The bloodbrain barrier is not totally impenetrable, but slows down the rate at which toxicants cross into brain tissue while allowing essential nutrients, including oxygen, to pass through -Type I astrocytes wrapping around brain capillaries as part of the BBB

Subchronic toxicity

-Subchronic toxicity results from repeated exposure for several weeks or months. This is a common human exposure pattern for some pharmaceuticals and environmental agents -Examples: - Ingestion of coumadin tablets (blood thinners) for several weeks as a treatment for venous thrombosis can cause internal bleeding. -Workplace exposure to lead over a period of several weeks can result in anemia

Superoxide dismutase

-Superoxide dismutase (SOD) specifically donates electrons to highly reactive superoxide anion radicals (O2.-) to form hydrogen peroxide (H2O2) and oxygen (O2). - Hydrogen peroxide is then broken down to water (H2O) and oxygen (O2) by another enzyme, catalase (CAT). - Free radicals must be reduced in biological tissues to prevent cellular damage, particularly lipid peroxidation.

Therapeutic Index

-The Therapeutic Index (TI) is used to compare the therapeutically effective dose to the toxic dose. -The TI is a statement of relative safety of a drug. It is the ratio of the dose producing toxicity to the dose needed to produce the desired therapeutic response. -The common method used to derive the TI is to use the 50% dose response values (TD50/ED50). TI = TD50/ED50 - For example, if the TD50 is 200 mg and the ED50 is 20 mg, the TI would be 10 (200/20). -A clinician would consider a drug safer if it had a TI of 10 than if it had a TI of 3. -Often the LD50 is used as the toxic dose (TD50).

geometric dose-response

-The basic principle of toxicology is that response varies proportionally to a geometric, not an arithmetic, increase in dose -this means that to test a substance that produces responses in a small proportion of animals at 1 to 2 mg/kg, a geometric dosing range (1, 2, 4, 8 and 16 mg/kg) would be used rather than an arithmetic range (1, 2, 3, 4 and 5 mg/kg) - Because of this, dose-response graphs are generally plotted with the response value on the y-axis and the logarithm of the dose on the x-axis

Barriers to disposition - Blood-Testis Barrier

-The blood-testis barrier protects the testes. -Small-molecular weight molecules (e.g. water, urea) can readily cross the blood-testis barrier while larger sized molecules (e.g. inulin) cannot (the degree of lipid solubility and ionization are also very important). -However, there are no known mechanisms or specialized barriers to prevent compounds from acting on the ovary.

detoxification

-The detoxification process typically converts lipid-soluble compounds to polar compounds to aid in their excretion in urine or feces. -The primary results of detoxification are: -the parent molecule is transformed into a more polar metabolite, often by the addition of ionizable groups - molecular weight and size are often increased - the excretion is facilitated, and hence elimination of the compound from the body is enhanced -The consequences of detoxification are: - the biological half-life is decreased -the duration of exposure is reduced -accumulation of the compound in the body is avoided -Detoxification will be most efficient when the parent compound can be altered to a metabolite that is hydrophilic, large and carries a charge (which will aid in excretion).

dosage

-The dosage is the most important and critical factor in determining if a substance will be an acute or a chronic toxicant. - Virtually all chemicals can be acute toxicants if sufficiently large doses are administered. -Often the toxic mechanisms and target organs are different for acute and chronic toxicity.

dose-response relationship

-The dose-response relationship is a fundamental and essential concept in toxicology. It correlates exposures and the spectrum of induced effects -Generally, the higher the dose, the more severe the response. The dose-response relationship is based on observed data from experimental animal, human clinical, or cell studies

form and innate chemical activity

-The form of a substance may have a profound impact on its toxicity especially for metallic elements. -The toxicity of mercury vapor differs greatly from methyl mercury. Another example is chromium. Cr3+ is relatively nontoxic whereas Cr6+ causes skin or nasal corrosion and lung cancer. -The innate chemical activity of substances also varies greatly. Some can quickly damage cells causing immediate cell death. Others slowly interfere only with a cell's function. -Hydrogen cyanide binds to cytochrome oxidase resulting in cellular hypoxia and rapid death; nicotine binds to cholinergic receptors in the CNS altering nerve conduction and inducing gradual onset of paralysis

The graph to the right shows a standard curve for measuring ATP using a luminometer - what enzyme is being used to drive this reaction?

-The graph shows a standard curve using luciferase to drive the following reaction: luciferin+ ATP+ O2-----> oxyluciferin + AMP+ pyrophosphate + CO2+ light --->= Mg2+ and luciferase -The luciferase assay is used to quantitatively determine ATP levels

Measuring mitochondrial membrane potential

-The inner mitochondrial membrane is normally maintained at a very negative potential in order to drive oxidative phosphorylation of ADP to form ATP. -Assays that measure loss of the negative inner membrane potential (depolarization) are commonly used to determine mitochondrial health, as an indicator of overall cell health. - Cells that have completely lost their inner mitochondrial membrane potential are considered to be dead.

Effective dose vs. Toxic dose

-The knowledge of the effective and toxic dose levels aides the toxicologist and clinician in determining the relative safety of pharmaceuticals. -As shown below, two dose-response curves are presented for the same drug, one for effectiveness and the other for toxicity. In this case, a dose that is 50-75% effective does not cause toxicity whereas a 90% effective dose may result in a small amount of toxicity.

Xenobiotic storage in liver and kidneys

-The liver is a storage site for some toxicants. -It has a large blood flow and hepatocytes contain proteins that bind to some chemicals, including toxicants. - As with the liver, the kidneys have a high blood flow, which preferentially exposes these organs to toxicants in high concentrations. - Storage in the kidneys is associated primarily with the cells of the nephron (the functional unit for urine formation).

How do toxic agents enter the body?

-The major routes (pathways) by which toxic agents gain access to the body are: -Gastrointestinal tract (ingestion) -Lungs (inhalation) -Skin (topical, percutaneous or dermal) and other parenteral routes. -Toxic agents generally produce the greatest effect and the most rapid response when introduced directly into the bloodstream (e.g. intravenously). An approximate descending order of effectiveness of the administration routes is: -Intravenous (most effective) -Inhalation -intraperitoneal -Subcutaneous -Intramuscular -Intradermal -Oral -Dermal (least effective) **note: "enteral" refers to all routes pertaining to the alimentary canal (sublingual, oral, rectal); "parenteral" involves all other routes

toxicity due to biochemical interactions

-The majority of toxic effects, especially due to xenobiotics, are due to specific biochemical interactions. -Examples: -Interference with a chemical that transmits a message across a neural synapse - Inhibition of the enzyme acetylcholinesterase by organophosphate pesticides - When one toxic chemical inhibits or replaces another essential chemical -Replacement of oxygen on the hemoglobin molecule with carbon monoxide - Some tissues have a great capacity for repair (most epithelial tissues). Others have limited or no capacity to regenerate and repair (nervous tissue). - Most organs have a functional reserve capacity so that they can continue to perform their body function although perhaps in somewhat diminished ability. Liver Kidney

depletion of protective factors

-The most common protective factor whose status is monitored during in vitro exposure to a potential toxicant is glutathione. -When glutathione becomes reduced due to a change in the overall redox status of the cell, the protection provided by glutathione becomes reduced or depleted, and the cell is now more vulnerable to damage due to oxidative stress.

measuring calcium dysregulation

-The most commonly used method is a fluorescent dye, such as fura-2 AM, that enters the cell and become trapped when the AM group is cleaved from the molecule by esterases within the cell. - Protocol: Cells are attached to glass coverslips, loaded with the dye, mounted on a fluorescent microscope, and excited with a highpowered light source (such as a xenon lamp) that excites the cells at a specific wavelength. The fura-2 will then emit light at one wavelength if it is bound to calcium, and at another if it is not bound to calcium, allowing for measurement of the [Ca2+]i within the cell. Scientists can then expose cells to known toxicants (positive controls) and unknown compounds (experimental condition) to determine whether the compound causes a brief (non-toxic) or prolonged (potentially toxic) increase in [Ca2+]i. -Other dyes for measuring changes in [Ca2+]i include fluo-3 and fluo4, among others. - In many pharmaceutical companies, the process of screening for a Ca2+ response has been automated using laboratory robots to handle everything from cell culture to exposure to compounds. -Calcium assays have the benefit of being rapid and relatively inexpensive; however, a prolonged increase in [Ca2+]i is not always an indicator of imminent cell death.

Phase 1 reactions

-The most important enzyme involved in biotransformation is cytochrome P-450 which catalyzes many Phase I reactions. -This enzyme is located primarily in the smooth endoplasmic reticulum (microsomal fraction) of the cell and is especially abundant in hepatocytes (stay tuned for more on cytochrome P450s when we discuss hepatotoxicity). -Cytochrome P450s primarily catalyze oxidation reactions and consist of many isoforms.

Barriers to disposition - Placental Barrier

-The placental barrier protects the developing fetus from toxicants distributed in the maternal circulation. -This barrier consists of several cell layers between the maternal and fetal circulatory vessels in the placenta. -Lipids in the cell membranes limit the diffusion of water-soluble toxicants. However, nutrients, gases, and wastes of the developing fetus can pass through the placental barrier. -As in the case of the blood-brain barrier, the placental barrier is not totally impenetrable.

Cellular response to injury primary event

-The primary events are the initial response to injury as a result of direct action of a toxic compound or one of its reactive metabolites -Common primary events include: -Lipid peroxidation -Enzyme inhibition - Oxidative stress -Covalent binding events - Changes in thiol status -Ischemia - A single toxic compound can have multiple primary events that contribute to cellular damage.

Factors that influence metabolism

-There are a number of factors that can affect both Phase I and Phase II reactions: - the availability of cofactors -the availability of co-substrates particular enzyme levels -especially important when comparing metabolic pathways across species - age - gender differences

changes in thiol status

-Thiol groups are sulfur-hydrogen functional groups that are found in the reactive sites of many enzymes, forming non-covalent bonds with substrates as part of normal enzyme functioning. -Thiol groups also regulate the redox status of glutathione, an intracellular protein that is important in protecting cells against oxidative stress. -Modification of thiol groups, either through covalent binding or alteration of redox status, can alter protein function. - Mercury binds irreversibly to thiol groups in a wide range of ion channels and enzymes, causing irreversible cellular damage.

Tissue Affinity

-Tissue affinity determines the degree of concentration of a toxicant. -In fact, some tissues have a higher affinity for specific chemicals and will accumulate a toxicant in great concentrations in spite of a rather low flow of blood. -For example, adipose tissue, which has a meager blood supply, concentrates lipid-soluble toxicants. Once deposited in these storage tissues, toxicants may remain for long periods of time, due to their solubility in the tissue and the relatively low blood flow. -During distribution, the passage of toxicants from capillaries into various tissues or organs is not uniform.

Tolerance

-Tolerance is a state of decreased responsiveness to a toxic effect of a chemical resulting from prior exposure to that chemical or to a structurally-related chemical. -There are two major mechanisms responsible for tolerance: one is due to a decreased amount of toxicant reaching the site where the toxic effect is produced (dispositional tolerance) and the other is due to a reduced responsiveness of a tissue to that chemical. - Examples are: -Carbon tetrachloride produces tolerance to itself by decreasing the formation of the reactive metabolite (trichloromethyl radical) that produces liver injury. -Cadmium produces tolerance by inducing metallothionein, a metal-binding protein; subsequent binding of cadmium to metallothionein rather than to critical macromolecules thus decreases its toxicity.

toxic damage to cells

-Toxic damage to cells and tissues can be transient and non-lethal or, in severe situations, the damage may cause death of the cells or tissues. -There are four main final outcomes to cellular or biochemical toxicity: - Complete repair -Incompletely repair - Death of the organism or the complete loss of a tissue or organ - Neoplasm or cancers -Toxicity is complex with many influencing factors; dosage is the most important. -Xenobiotics cause many types of toxicity by a variety of mechanisms. -Some chemicals are themselves toxic. - Others must be metabolized (chemically changed within the body) before they cause toxicity. - Many xenobiotics distribute in the body and often affect only specific target organs. - Others, however, can damage any cell or tissue that they contact.

Species

-Toxic responses can vary substantially depending on the species. -Most species differences are attributable to differences in metabolism. - Others may be due to anatomical or physiological differences. - Rats cannot vomit and expel toxicants before they are absorbed or cause severe irritation, whereas humans and dogs are capable of vomiting. - Selective toxicity refers to species differences in toxicity between two species simultaneously exposed. This is the basis for the effectiveness of pesticides and drugs. - An insecticide is lethal to insects but relatively nontoxic to animals; antibiotics are selectively toxic to microorganisms while virtually nontoxic to humans.

systemic toxicity

-Toxic substances may be systemic toxicants or organ toxicants. - A systemic toxicant is one that affects the entire body or many organs rather than a specific site. -Potassium cyanide -Toxic effects are generally categorized according to the site of the toxic effect. -Target organ is the specific site of action - Systemic toxicity occurs when toxicity is at multiple sites

Target Organ Toxicity

-Toxicants may affect only specific tissues or organs while not producing damage to the body as a whole. -These specific sites are known as the target organs or target tissues. - For example, benzene is a specific organ toxicant in that it is primarily toxic to the blood-forming tissues (hematopoietic system). -Lead is also a specific organ toxicant; however, it has three target organs (central nervous system, kidney and hematopoietic system).

IP, IM, IV

-Toxicity may also be assessed by direct injection of the substance, using a syringe and needle. -Intraperitoneal (ip) injections are made into the body cavity -Intramuscular (im) injections are placed into a large muscle of the hind leg; subcutaneous (sc) injections are placed just beneath the skin -Intravenous (iv) injections are made directly into a large vein.

Glutathione Assays

-Unlike calcium assays, which use intact, viable cells, glutathione assays require the cells to be lysed so that the assay reagents can interact with the intracellular glutathione. -For this reason, glutathione assays are a good measure of the redox status of a cell, but cannot be used to measure changes in redox status in a cell in real time, and, like calcium assays, do not provide a direct measure of cell viability. -Glutathione kits are relatively inexpensive, require only a spectrophotometer for reading absorbance, and can be performed rapidly, especially when in an automated laboratory.

Acute toxicity

-acute toxicity occurs almost immediately (hours/days) after an exposure. - An acute exposure is usually a single dose or a series of doses received within a 24 hour period. - Death is a major concern in cases of acute exposures. - Examples: - In 1989, 5,000 people died and 30,000 were permanently disabled due to exposure to methyl isocyanate from an industrial accident in Bhopal, India. - Many people die each year from inhaling carbon monoxide released from faulty heaters; non-lethal acute effects may also occur (e.g. convulsions and respiratory irritation).

History

-dates back to the earliest humans, who used animal venoms and plant extracts (poisons) for hunting and warfare. -By 1500 BC, written recordings (such as the Ebers papyrus) indicated that hemlock, opium, arrow poisons, and certain metals (such as lead, copper and antimony) were used to poison enemies or for state executions (and assassinations). -With time, poisons became widely used and with great sophistication -history is full of poisonings -By the time of the Renaissance and Age of Enlightenment, certain concepts fundamental to toxicology began to take shape -Noteworthy in this regard were the studies of Paracelsus (~1500AD) and Orfila (~1800 AD)

lethal dose

-dose at which a certain percentage of individuals are expected to die -A common dose estimate for acute toxicity is the LD50 (Lethal Dose 50%). This is a statistically derived dose at which 50% of the individuals are expected to die. The figure illustrates how an LD50 (20 mg for this particular compound) is derived -Other dose estimates also may be used. LD0 represents the dose at which no individuals are expected to die whereas LD10 refers to the dose at which 10% of the individuals are expected to die. -For inhalation toxicity, air concentrations are used for exposure values. Thus, the LC50 is utilized which stands for Lethal Concentration 50% (the calculated concentration of a gas lethal to 50% of a group). Occasionally LC0 and LC10 are also used as well.

Humane animal care

-handling and treatment of animals must be humane and must be the same for all animals in the study, whether they are in treatment or control groups. -Animals must be housed in clean, comfortable conditions with access to adequate food and water. -Typically, animals are housed in conventional, box-type cages, although in some cases specialized cages (such as metabolism cages) may be used

Caspases & Death Effector Domains

-synthesized as inactive zymogens called procaspases -Domain = stretch of protein that has a specific function - Initiator caspases respond to cell death signals: - Long prodomains (are prior to active portion of protein - cleaved or otherwise altered in zymogen to activate protein) - Death Domains (DD) = protein interaction domain similar to DED (below) = pro-cell-death signal - Caspase Recruitment Domains (CARD) = allows caspses to interact with other caspses - Executioner (effector) caspases trigger apoptosis: - Activated by initiator caspases - Short prodomains - Death Effector Domains (DED) = regulates caspase activation to promote cell death

Changes in membrane structure & permeability

-the ability of the plasma membrane to form a protective barrier depends on the maintenance of its impermeable structure. - Similarly, intracellular organelles are enveloped by membranes that help retain function. - When membrane structure is compromised, cellular integrity and function is degraded and often leads to cell death. -When viewed through the microscope, membrane damage can often be visualized as mitochondrial swelling and disruption of the endoplasmic reticulum network. - Altered plasma membrane permeability is the basis for several cell viability assays, since loss of plasma membrane integrity is a strong indicator of cell death.

Dose

-the amount of a substance administered at one time -However, other parameters are needed to characterize the exposure to xenobiotics. The most important are: -the number of doses -the dosing frequency -total time period of the treatment Some examples of doses: -650 mg acetaminophen as a single dose -500 mg penicillin every 8 hours for 10 days -10 mg atrazine per day for 90 days

Balance between detoxification & toxification

-the balance between detoxification and toxification can be affected by: relative rates of the toxification and detoxification pathways; these will be influenced by the availability of enzymes and the kinetic parameters of these enzymes availability of cofactors protective mechanisms dose saturability of the metabolic pathway genetic variation induction or inhibition of the enzymes involved species or strain of animal tissue differences in enzyme and enzyme isoform patterns diet age disease state sex

The dose-response relationship is based on certain assumptions

-the effect is due to the chemical administered -the magnitude of response is related to the dose -a molecular target exists with which the chemical interacts to initiate a response -the response is related to the concentration of the chemical at the target site -the concentration at the target site is due to the amount administered -there is a quantifiable way to measure the response Knowledge of the dose-response relationship: -establishes the threshold (lowest dose where an induced effect occurs) -determines the slope of the dose response (the rate at which injury builds up)

Absorption from the GI tract

-toxicants must be absorbed to cause systemic effects -absorption may be through passive diffusion across membranes or active transport -Very few absorbed by active transport -Absorption is favored by: Lipid solubility, Size, Concentration, Surface area, Blood flow

Bioactivation

-toxication (toxification) occurs when the biotransformation of a xenobiotic results in metabolites or intermediates that are more toxic than the parent compound. -Bioactivation may be the result of Phase I, II or III reactions although Phase I reactions are commonly involved. -The intermediates or metabolites responsible for the toxicity may be chemically reactive or stable. -When the metabolic process produces a metabolite which is chemically reactive, the process is known as metabolic activation or bioactivation.

Additivity

-when the combined effect of two chemicals is equal to the sum of the effects of each agent given alone -Additivity is the most common type of drug interaction. -Examples of chemical or drug additivity reactions are: -Two central nervous system (CNS) depressants taken at the same time, a tranquilizer and alcohol, often cause depression equal to the sum of that caused by each drug. -Organophosphate insecticides interfere with nerve conduction. The toxicity of the combination of two organophosphate insecticides is equal to the sum of the toxicity of each. -Chlorinated insecticides and halogenated solvents both produce liver toxicity. The hepatotoxicity of an insecticide formulation containing both is equivalent to the sum of the hepatotoxicity of each.

Antagonism

-when two chemicals administered together interfere with each other's actions or one interferes with the action of the other; the exposure to one chemical results in a reduction in the effect of the other chemical. -often a desirable effect in toxicology and is the basis for most antidotes