World-Class Study Notes on Food Toxicology

Fundamentals of Food Toxicology

• Toxicology: The study of harmful interactions between chemical substances and living beings, as well as the probabilities of their occurrence in the past or future.

• Food Toxicology: Knowledge regarding the presence of harmful substances in food and the prevention of consuming quantities that put the health of the consumer at risk.

• Xenobiotic: A foreign substance that enters the organism from the external environment.

• Toxic: Any substance capable of producing harmful interactions with living organisms.

• Poison: Intoxications considered as homicidal or suicidal; accidental intoxications are never classified as poisonings.

• Factors of Adverse Effects: The influence of toxics and/or toxic metabolites on biological systems depends on:

  • Target Organ (Target): The specific organ where the toxic exerts its effect (not necessarily where it is stored).

  • Adequate Concentration: The level required to cause harm.

  • Sufficient Time: The duration of exposure required for the effect to manifest.

• $LD_{50}$ (Lethal Dose 50%): Represents the dose that causes death in $50\%$ of the exposed population. Used specifically for lethal components.

• Toxicity Relationship: The lower the $LD_{50}$, the higher the toxicity, as a smaller dose is needed to be lethal.

Biological and Exposure Variations

• Inter-species Variation: The toxic effect varies depending on the specific living organism (e.g., humans vs. rats).

• Intra-species Variation: Differences within the same species based on sex, age, immune defenses, and allergies.

• Comparative Toxicology: Studies which animal model should be used to extrapolate results to humans.

• Absorption Routes: Velocity and penetration power vary by route. A toxic must cross barriers to produce an effect; it may be lethal via one route but not another.

• Types of Intoxication:

  • Acute Intoxication: A single dose that produces effects within a few hours. Results in death or total/partial recovery, with or without persistent lesions.

  • Subacute Intoxication: Pathology appearing in a short time (days or weeks) from sub-doses administered over a few days. Visible disorders may not appear initially but manifest shortly after.

  • Chronic Intoxication: Pathology resulting from repeated absorption of doses that do not cause acute effects. It can take months or years to express. Involves accumulation of the xenobiotic in specific organs or tissues, often resulting in irreversible damage.

• Elimination: Toxins are primarily eliminated via Urine (hydrophilic and ionized compounds). Secondary routes include Feces, Bile, Expired Air (volatile compounds), Milk, Saliva, and Sweat.

Toxicological Metrics and Definitions

• Dose: The absolute amount of a substance entering the organism (expressed in $mg$, $g$, or $ml$).

• Dosage: The amount of toxic received relative to body weight (e.g., $mg/Kg$).

• Concentration: Amount of substance in a specific medium where the target organism is found.

• $LC_{50}$ (Lethal Concentration 50%): The amount of toxic in the environment (air, water, food) that kills $50\%$ of the population. Often used for atmospheric or volatile xenobiotics via the pulmonary route (expressed in $ppm$ or $mg/Kg$).

• $TD_{50}$ (Toxic Dose 50%): The dose that causes a specific toxic effect (not necessarily death) in $50\%$ of animal subjects.

• $ED_{50}$ (Effective Dose 50%): The dose that causes the desired/sought effect in $50\%$ of the population.

Dose-Response Relationship and Safety Indices

• Relationship Logic: The intensity of the response is proportional to the exposed dose.

• Experimental Requirements: Establishing a dose-response curve requires:

  • Selection of monitored response type (quantitative).

  • Definition of the test organism (biological system/target organ).

  • Experiment duration.

  • Dose series: single dose (acute), repetitive short-term (subacute), repetitive long-term (chronic).

  • Choice of administration route.

• Threshold: The value where a response to the toxic begins to be observed.

• Safety Index (IS): Determined by the overlap between the highest effective doses and the lowest lethal doses.

  • IS < 1: Indicates overlap between lethal and therapeutic doses (only acceptable for anti-cancer or HIV drugs).

  • IS >>> 1: Indicates a safer drug.

• NO(A)EL / DSEO (No Observable Adverse Effect Level): The maximum dose of a substance that causes no distinguishable adverse effect in experimental animals during toxicity studies.

• LO(A)EL (Lowest Observed Adverse Effect Level): The lowest concentration causing a distinguishable adverse change compared to a normal (control) organism.

Regulatory Limits and Safety Factors

• IDA / ADI (Acceptable Daily Intake): The amount that can be consumed daily throughout life without harm, measured in $mg/Kg/day$.

• Calculation: $IDA = \frac{DSEO}{FS}$

• Safety Factor ($FS$ / $sf$): A dimensionless measure to account for inter-species and intra-species extrapolation.

  • $FS = 10$: When DSEO is from the same species.

  • $FS = 100$: When DSEO is from a different species.

  • FS > 100: When data is inconclusive or variability is high.

• High FS Implications: Significant reduction of DSEO to calculate IDA; indicates high uncertainty, extreme precaution, or potential high toxicity at low doses in humans.

• LMR (Maximum Residue Limit): Primarily used for pesticides.

  • Formula: $LMR = \frac{1000 \times W}{a} \times DDA$

  • $W$: Weight of the individual ($Kg$).

  • $a$: Average daily consumption of the food ($g/day$).

  • $DDA$: IDA ($mg/Kg \, p.c.-day$).

  • Units: $ppm$ ($mg/Kg$ of food).

• Molecular Interactions:

  • Additive Effect: $3 + 1 = 4$

  • Synergistic Effect: $3 + 1 = 9$

  • Potentiation: $0 + 3 = 10$

  • Antagonism: $3 + 2 = 0$

Absorption and Membrane Dynamics

• Cellular Membranes: Toxic chemicals cross membranes based on composition.

  • Hydrophobic substances: Cross easily via passive diffusion.

  • Ionizable substances: Cross better in their non-ionized (liposoluble) form.

  • Hydrophilic substances: Require transport proteins.

• pH and Ionization Relationship:

  • Formula: $pKa - pH = \log(\frac{\text{non-ionized}}{\text{ionized}})$

  • Weak Acids (e.g., Benzoic Acid, $pKa = 4$): In the stomach ($pH = 2$), the ratio is $100:1$ (non-ionized), leading to better absorption. In the intestine ($pH = 6$), the ratio is $1:100$ (ionized), reducing absorption.

  • Weak Bases (e.g., Aniline, $pKa = 5$): In the stomach ($pH = 2$), the ratio is $1:1000$ (ionized), hindering absorption. In the intestine ($pH = 6$), the ratio is $1:10$ (non-ionized), favoring absorption.

• Gastrointestinal Tract (TGI): Key for food toxicology. Absorption depends on pH, digestive enzymes, intestinal flora (determines half-life), and motility (residence time).

Distribution and Biotransformation

• Equilibrium: Stored toxic is in equilibrium with free toxic in the plasma; as it is metabolized, stored toxic is released, resulting in a long half-life.

• Biotransformation: Structural modifications, primarily in the liver, to generate less toxic, less liposoluble (more polar) compounds for excretion.

• Phase I Reactions (Functionalization):

  • Oxidation: Most important. Uses the Cytochrome P-450 enzyme system in the liver. Requires $NADPH$ as electron donor and $O_2$ as oxidant.

  • Others: Reduction (azo dyes to amines), Hydrolysis (esterases, amidases).

• Phase II Reactions (Conjugation): Requires $ATP$ and cofactors. Purpose: Increase polarity and decrease lipid/water partition coefficient.

  • Glucuronidation: Uses UDP-glucuronyltransferase and glucuronic acid. Main conjugation process.

  • Sulfation: Uses Sulfotransferase and $PAPS$. Detoxifying process.

  • Glutathione Conjugation: Uses glutathione-transferases; excreted as N-acetylcysteine conjugates.

• Bioactivation: An exception where biotransformation increases activity and toxicity (e.g., N-hydroxyacetylaminofluorene becoming a carcinogen).

• Enterohepatic Cycle: Compounds excreted via bile into the intestine may be reabsorbed. Poor liver function can prolong half-life and increase toxicity via this cycle.

Analytical Methods in Food Toxicology

• Sampling: Must be representative; includes specific units and subunits of a lot.

• Analysis Phases: Solvent Extraction ($low \, b.p.$, non-toxic), Purification, and Final Analysis.

• TLC (Thin Layer Chromatography): Qualitative method. Compares sample to a control under $UV$ light.

• ELISA (Enzyme-Linked Immunosorbent Assay): Based on antigen-antibody reactions.

  • Sandwich ELISA: Primary antibody captures antigen; secondary enzyme-marked antibody detects it. More color = higher concentration.

  • Competitive ELISA: Higher antigen in the sample = lower signal (fewer antibodies available to bind).

• PCR (Polymerase Chain Reaction): Exponential DNA replication.

  • Cycles: $94^{\circ}C$ (Denaturation), $52^{\circ}C$ (Annealing of primers), $72^{\circ}C$ (Duplication by DNA polymerase).

  • IAC (Internal Application Control): A slightly heavier molecule used to verify if the PCR worked. If IAC is not visible, the test is invalid.

Toxicity Testing Levels and Protocols

• Testing Levels:

  • Level I: Bacteriological/Viral.

  • Level II: Cell Culture.

  • Level III: Whole Organism.

  • Level IV: Epidemiological.

• Mutagenesis Tests:

  • Ames Test: Uses Salmonella typhimurium with blocked histidine synthesis and rat liver homogenate ($S9$). Double the growth (revertants) compared to control indicates toxicity.

  • Host-Mediated Assay: Bacteria are injected into a live animal; the animal is treated with the chemical; bacteria are extracted to see if metabolites caused mutations.

  • Dominant Lethal Test: Treated males mate with untreated females; zygote death or fetal abnormalities indicate dominant lethal mutations.

• Subchronic Studies: 90 days in mice to evaluate accumulative effects (biochemistry, weight, feces, pathology).

• Teratogenesis: Abnormalities in zygote development. Organogenesis is the most sensitive phase (Days $10-12$ in rats).

• The 3Rs of Animal Research:

  • Reduction: Use fewer animals.

  • Replacement: Use cultures or microorganisms where possible.

  • Refinement: Reduce pain/discomfort via enrichment and analgesics.

• Bioassays:

  • Artemia salina: Larvae mortality measured over $24 \, h$. Scale: $0-9\%$ (Non-toxic), $10-49\%$ (Slightly toxic), $50-89\%$ (Toxic), >90\% (Very toxic).

Specific Toxins: Marine and Biogenic Amines

• Histamine: Formed by bacterial decarboxylation of free histidine ($pH$ acidic, $T = 5-60^{\circ}C$). Indicator of fish deterioration. Thermostable.

  • Symptoms: Ranges from light (pruritus, facial flushing) to severe (urticaria, respiratory distress, anxiety).

  • Limits: Average < $100 \, ppm$, max $200 \, ppm$ for any sample.

• Water-Soluble Marine Biotoxins:

  • Domoic Acid (DA): Produced by Pseudonitzschia. Amnesic Shellfish Poisoning (ASP). Destroys hippocampal neurons by Ca++ influx.

  • Saxitoxins (STX): Produced by Gonyaulax catenella. Paralytic Shellfish Poisoning (PSP). Blocks Na+ channels, causing respiratory paralysis. Stable to heat (retains $50\%$ after $116^{\circ}C$ for $4 \, h$).

  • Tetrodotoxin: From pufferfish; produced by Alteromonas sp. bacteria. Inhibits Na+ channels. $1000\text{x}$ more toxic than cyanide.

• Lipophilic Marine Biotoxins:

  • Ciguatoxins: Results from biotransformation of gambiertoxins in large fish (Gambierdiscus toxicus). Opens Na+ channels, causing sustained depolarization.

  • Okadaic Acid (OA): Diarrhetic Shellfish Poisoning (DSP). Causes vomiting and diarrhea.

Natural Plant Toxins

• Glucosinolates: Found in seeds (mustard) and Cruciferae. Inhibits iodine absorption, causing goiter (Bocio). Inactivated at $90^{\circ}C$ for $15 \, min$.

• Cyanogenic Glycosides: Release Cyanide ($CN^-$) via enzyme action. Inhibits Cytochrome Oxidase, blocking cellular respiration. Found in cassava and bitter almonds (Amygdalin).

• Favism: Anemia hemolytica caused by broad beans in individuals with a genetic deficiency in G6PD. Lack of G6PD prevents $NADPH$ and reduced glutathione production.

• Latirismo: Linked to Lathyrus sativus (almorta). Causes neuro- and osteolatirismo.

• Solanine/Chaconina: Glycoalkaloids in immature potatoes (skin/sprouts). Inhibits cholinesterase, disrupting nerve impulses. Termoresistant.

• Lectins: Glycoproteins that agglutinate blood. Found in ricin and legumes. Destroy intestinal epitelio.

Food Additives and Preservation

• Definition: Intentional ingredients added to modify characteristics (sensory, physical, chemical) during processing or storage, without nutritional purpose.

• Positive Lists: Additives must be on official lists (safety and efficacy evaluated) to be used.

• Preservatives:

  • Benzoic Acid: Active at low pH ($2.4 - 4.0$) in non-dissociated form ($IDA: 0-5 \, mg/Kg$).

  • Sorbic Acid: Metabolized like a fatty acid ($IDA: 25 \, mg/Kg$). Active in weakly acidic foods.

  • Parabens: Active at neutral pH; provide phenolic odor; excreted rapidly.

  • Nitrites/Nitrates: Used in cured meats to prevent Clostridium botulinum. Can form carcinogenic Nitrosamines in the stomach and cause Methemoglobinemia (Blue Baby Syndrome) by preventing oxygen binding in hemoglobin ($LD_{50}: 30-35 \, g/Kg$ in humans).

• Sweeteners:

  • Cyclamate: $30-35\text{x}$ sweeter than sugar. Banned in some countries due to bladder carcinoma risk in animal studies ($IDA: 11 \, mg/Kg$).

  • Saccharin: $300\text{x}$ sweeter. Rat studies only showed tumors at extremely high doses.

  • Aspartame: Composed of aspartic acid and phenylalanine methyl ester. Dangerous for those with Phenylketonuria. ($IDA: 40 \, mg/Kg$).

• Bromate of Potassium: Former bread improver (1914). Banned in Argentina (1993) due to carcinogenicity, though illegal use still occurs.