Introduction to Toxicology

• Toxicology is a multidisciplinary science studying chemicals' adverse effects on biological systems
• Modern toxicology consists of descriptive, mechanistic, and regulatory subdisciplines

Toxicology Subdisciplines

• Descriptive toxicology: Involves toxicity testing of chemicals
• Mechanistic toxicology: Examines biochemical processes by which identified toxicants impact organisms
• Regulatory toxicology: Assesses data from descriptive and mechanistic toxicology to determine legal uses and ecosystem risks

Hazardous Waste

• Increasing industries and global population leads to accumulation of waste
• Hazardous waste: Waste that poses a danger of morbidity or mortality to organisms due to biological, chemical, physical characteristics, quantity, or concentration
• In the U.S. alone, over 4 billion tons of waste are generated each year from mining, agriculture, industry, and city sewage

Toxicological Concepts

• Toxicology: Study of poisons or toxicants
• Four distinct types of toxicity classify the duration and location of the poisonous state

Types of Toxicity

• Acute toxicity: Sudden onset of symptoms, lasting less than 24 hours
• Cellular damage from acute toxicity is usually reversible
• Chronic toxicity: Continuous symptoms from irreversible cellular changes
• Local toxicity: Symptoms restricted to the initial exposure site of the toxicant
• Systemic toxicity: Adverse effects occur far from the initial exposure site
• Repeated exposure to vapors or liquids can cause irreversible chronic and systemic toxicity
• Kidney and liver damage, and CNS depression are pathologies associated with chronic toxicity, which can be life-threatening

Toxicity Classification by Timing

• Immediate toxicity: Symptoms appear rapidly (within seconds or minutes) after exposure
• Delayed toxicity: Symptoms appear years after exposure
• Delayed toxicity makes it difficult to establish cause-and-effect relationships
• Characterizing toxicity (acute, local, immediate, chronic, systemic, delayed) helps understand effects of toxicants on organisms

Toxicokinetics and Toxicodynamics

• Toxicokinetics: Study of 5 time-dependent processes related to toxicants interacting with living organisms
  • Absorption: How toxicants enter the organism
  • Distribution: How toxicants travel within the organism
  • Storage: How tissues preferentially harbor toxicants
  • Biotransformation: How toxicants are altered or detoxified
  • Elimination: How toxicants are removed
• Toxicodynamics: Examines mechanisms by which toxicants produce unique cellular effects

Cellular Effects of Toxicants

• Can involve plasma membrane alterations
• Impact organelles
• Impact the nucleus
• Effects on cytoplasm
• Effects on enzyme systems
• Effects on biosynthetic pathways
• Effects on development
• Effects reproduction
• The degree of cellular injury depends on exposure duration and toxicokinetic properties

Toxicity Testing

• Four stages of toxicity testing
  • Select test organisms (plants or animals) like algae, bacteria, mice, rats, rabbits, or nonhuman primates
  • Responses (end effects) to be observed and recorded
    • Changes in cell numbers for a bacterial colony
    • Absence/presence of biochemicals from cultured cells
    • Cell morphology changes
    • Number of tumors
    • Alterations in sleep
    • Changes in organism growth/development
    • Death of experimental in vivo organism
  • Select duration of exposure (seconds to years)
  • Select doses to be tested
    • Expressed as mg of substance per kg of body weight for in vivo studies
    • Expressed as mg of substance per mL for in vitro studies

Dose Selection

• Logarithmic dose sequences are preferred over linear to maximize tested dose ranges
• Logarithmic also minimizes overlooking the response threshold or minimum effective dose

Dose-Response Relationship

• Exists when a consistent mathematical relationship describes the proportion of organisms responding to a specific dose for a given exposure
• Graph the dose-response data for environmental toxicologists to determine relationships
• Horizontal axis (X-axis/abscissa) represents dose in mg/kg
• Vertical axis (Y-axis/ordinate) represents in vivo or in vitro response

Dose-Response Graphs

• Response axis may represent frequency or cumulative response
• Frequency dose-response: Plots percentage of organisms responding to a given dose
• These graphs have a bell-shaped appearance
• Cumulative dose-response: Sums responses from lower to higher doses
• Line on graphs look sigmoidal
• Test organisms show a bell-shaped distribution

Distribution of Test Organism Responses

• Few organisms respond at very low doses ("supersensitive")
• Few organisms respond to high doses only ("resistant")
• Resistant and supersensitive organisms are outliers graphed at the graph sides or sigmoidal line beginning/end
• Determine outliers' distance from the mean through standard deviation (SD)
• Plus or minus one SD: 67% of test organism responses
• Plus or minus two SD accounts: 95% of test organism responses
• Plus or minus three SD accounts: 99% of test organism responses
• Wide range of doses elicits a response if toxicant has a large SD (versus one with a small SD)

Sigmoidal Line Characteristics on cumulative dose-response graphs

• Threshold dose, where the first test organism responds, is at the graph's left side beginning
• Subthreshold doses, to the left of this point, elicit no response
• Terms include No Observable Effects Level (NOEL), No Observable Adverse Effect Level (NOAEL), Suggested No Adverse Response Level (SNARL), Lowest Observable Effect Limit (LOEL), Threshold Limit Value (TLV)
• The sigmoidal line straightens at progressively higher doses
• The majority of test organisms exhibiting a response are in this second region of graph
• The cumulative 50% level represents the mean response
• Right side: Cumulative dose-response graphs curve again and become horizontal; remaining test organisms exhibit predetermined end effect
• This portion is called the "ceiling effect"
• A dose increase produces little or no response increase

Cumulative Dose-Response: 100% Level

• Cumulative 100% level: Where graph stops
• Characterize dosages as effective, toxic, or lethal based on response
• Eeffective dose (ED): A desirable response is observed
• Toxic dose (TD): The dose at which toxicity appears
• Lethal dose (LD): The dose results in death
• Lethality can be ED
• Therapeutic dose: Should be ED, not TD
• Numerical subscript denotes cumulative percentage showing the predetermined response; ED50, TD50, and LD50 are the values at which 50% of test organisms exhibit those responses
• 50% is often used, but other values are used with ED99, TD10, or LD01

Relative Toxicity Determination

• Smaller ED50, TD50, or LD50 values indicate a more potent toxicant
• Potency: Responses at lower doses
• Potency is comparative using percentages
• Efficacy: Toxicant sustains the dose-response over a broader range
• Some toxicants produce 100% response over a short range
• Some sustain responses even at higher doses

Mixed/Reversed Toxicity Relationship

• Sigmoidal lines can intersect
• This occurs if one toxicant isn't consistently potent
• This is when dose-response curves cross
• LD10: Toxicant A is higher than toxicant B, but LD50 for toxicant A is lower than toxicant B

Margin of Safety

• Expresses range magnitude between the non-effective or effective dose and lethal dose
• Determined by result with two studies that had an ED and LD
• The ED study indicates the dose at which the reversible toxic signs are produced, and establishes a threshold dose
• TD or LD studies establish the doses that may be irreversible and/or lethal
• A ratio between selected LD and ED values express safety margin, like LD01/ED99 or TD50/ED50
• When the ratio is larger = greater margin of safety is indicated

Acceptable Exposures

• These ratios are useful in determining acceptable exposures to environment
• Large LD01 or ED99 is useful for pharmaceuticals
• Implies therapeutic value from low doses (relative to lethal)
• Important with overdose potency

Nonnutritional Toxic Substances

• Dose-response curves of some nonnutritional toxic substances may exhibit non-traditional shapes
• Agents induce stimulatory effects at low doses but at higher doses they may be adverse - hormesis

Toxic Responses: Variation

• Toxicity varies by exposure duration/location and species response

Four Types of Poisons or Toxicants

• Acute toxicity: Sudden, less than 24 hours in duration
• Acute cellular damage is reversible
• Chronic toxicity: Prolonged, continuous duration
• Chronic - irreversible cellular changes occur
• Local toxicity: Toxicity restricted to the initial exposure area
• Systemic toxicity: Toxicity away from initial exposure site

Responses:

• Immediate toxicity has symptoms appearing rapidly
• Delayed toxicity onset is much later, perhaps years later
• Delayed toxicity makes cause and effect more difficult to identify

Toxicants: Entrance

• Toxicants enter body through absorption
• Ingested/inhaled materials are outside body until they pass barriers of the gastrointestinal tract or respiratory system
• Irritation can occur, but toxicants exert effects on internal organs
• Absorption rate varies by chemical and exposure route

Absorption Dose vs Exposure Dose

• Only a fraction of an exposure by skin, oral, or respiratory route is actually absorbed
• Exposure dose is the same as absorbed dose for injected/ implanted substances
• Route, substance, and chemical property all effect Xenobiotic absorption

Route-Specific Absorption

• Xenobiotics are ranked for hazard based on exposure route
• Substances aren't toxic, but exposure may result in high toxicity

Xenobiotic Entry Routes

• Gastrointestinal (GI) tract: Environmental and pharmaceutical contaminants
• Respiratory tract: Environmental/occupational exposure contaminants and air contaminants
• Skin: Environmental/occupational exposures and consumer/pharmaceutical products

Cell Membrane Details

• Cell membranes (plasma membranes) surround all the body's cells
• They're phospholipid bilayers
• Each has a phosphate head (hydrophilic and polar)
• Each has a lipid tail (lipophilic)

Xenobiotic and Cell Walls

• To enter, move, and leave an organism, xenobiotics must cross cell membranes (wall)
• Cell membrane is a body defense that prevents foreign invaders

Xenobiotics and Solid Tissues

• Solid cells (skin/mucous membrane) are tightly packed
• Blocks intercellular flow of substances
• Xenobiotics must penetrate cell walls to move between solid tissues
• Several movements across the membrane must occur to change physical location after passing between cells
• Barriers

Movement of Toxicants

• Some toxicants move easily while others struggle to move across cell membranes
• Passive or facilitated transfer
• Passive transfer: Simple diffusion/osmotic filtration, no energy/assistance needed
• Active transport is also possible, with assistance and energy needed

Passive Transfer Details

• Most common xenobiotic transfer method
• Affected by membrane pores, or hydrophobic interiors
• The two major factors are substance concentration in solution and the size of openings that the diffusing agent must negotiate

Substance Attributes that Affect Passive Transfer

• Lipid solubility
• Molecular size
• Degree of ionization

Diffusion, Molecular Size and Membrane Thickness

• High lipid soluble substances diffuse via the phospholipid membrane
• Small, water-soluble substances move through pores while intracellular fluid flows
• Larger water-soluble molecules generally can't pass though the small pores, though some may slowly diffuse via lipid portions
• Most aqueous pores measure ~4 Å, permitting chemicals with molecular weights of 100-200 to pass
• Exceptions are capillary/kidney glomeruli molecules, which have pores of ~40 Å that allow compounds with molecular weight up to ~50,000
• Highly ionized compounds generally have low lipid and thus are not able to pass through the membrane easily

Facilitated Diffusion:

• Similar to simple diffusion
• No energy is required
• Follows diffusion, but has carrier-mediated transport (specialized proteins)
• Can move larger molecules across membrane