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● Intracellular receptors are found in either the cytosol or the nucleus. The intracellular recep-tors are typically hormone receprecep-tors with natural ligands including androgens, oestro-gens, glucocorticoids and mineralocorticoids.

● An intracellular receptor often mentioned in connection with toxic effects is the so-called aryl hydrocarbon (Ah) receptor. Ah is a cytosolic transcription factor that is normally inactive, bound to several co-chaperones.

Upon ligand binding the chaperones dissoci-ate, resulting in Ah translocating into the nucleus and leading to changes in gene tran-scription. Some chemicals including the very toxic dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) interact with this receptor.

ENZYMES. Many toxic compounds influence the organism by inhibiting enzymes. Such inhibition may be non-specific as is the background for a number of the effects of toxic heavy metals such as lead and mercury. The general mechanism in this case is a high affinity to thiol (–SH) groups present in all enzymes. Even so, some enzymes are more susceptible to inhibition by these metals than oth-ers, an example being d-aminolevulinic acid dehy-drase, the activity of which in erythrocytes can be used as an early indicator of lead poisoning.

Inhibition may also be more specific; examples are the following:

● Inhibition of acetylcholinesterase by either organophosphates (which can be insecticides or

warfare gases) or carbamate insecticides. In the latter case the inhibition is reversible, which is not always the case for the inhibition caused by organophosphates.

● Dinitrophenols have earlier been used inten-sively as herbicides. An example is the com-pound 4,6-dinitro-o-cresol (DNOC). Like other dinitrophenols, DNOC increases the oxidative metabolism and heat production by direct cellular reaction. DNOC affects enzyme systems by inhibiting the formation of ATP and by blocking oxidative phosphorylation.

● Inhibition of an enzyme may also happen after several stages of transformation of the original compound absorbed. The key example of this situation, which is also called lethal synthesis, is the inhibition of the citric acid cycle enzyme aconitase by fluorocitric acid, a reaction that blocks energy production. The fluorocitric acid is formed if an individual is exposed to either synthetic fluoroacetic acid or to the same com-pound found as a natural toxic plant constitu-ent in ‘gifblaar’, a small shrub (Dichapetalum cymosum) well known as a livestock poison in South Africa. But we do not stop here. Actually some other species of Dichapetalum, such as D. toxicarium, contain in its seeds long-chain fluorine fatty acids such as w-fluoro-oleic acid and w-fluoro-palmitic acid. After ingestion these fluorine fatty acids are b-oxidized to form the aforementioned fluoroacetic acid. This in turn enters the citric acid cycle where it is metabolized into fluorocitric acid. This cannot be metabolized further since aconitase does not accept this compound instead of its natural substrate citric acid. The cycle is blocked.

CARRIERS. Here we think first of all of hae-moglobin, the iron-containing protein capable of transporting oxygen and carbon dioxide in the blood. This pigment of erythrocytes, formed by developing erythrocytes in the bone marrow, has an iron haem moiety and four different polypeptide globin chains that contain between 141 and 146 amino acids. Haemoglobin A is the normal adult haemoglobin and haemoglobin F is fetal haemo-globin. Haemoglobin F is the main oxygen-trans-port protein in the fetus during the last 7 months of development in the uterus and in the newborn until around 6 months. Functionally, fetal haemoglobin differs most from adult haemoglobin in that it is

able to bind oxygen with greater affinity than the adult form, giving the developing fetus better access to oxygen from the mother’s bloodstream.

The iron haem moiety binds reversibly to oxygen to form oxyhaemoglobin and a globin moiety binds reversibly to carbon dioxide to form carbamino-haemoglobin. Under normal conditions around 97% of the oxygen is transported bound to haemo-globin, whereas only 3% is dissolved in the plasma.

Only about 20% of the blood carbon dioxide is transported bound to haemoglobin. Oxygen trans-port can be impaired by interaction with a number of different toxic compounds.

● Carbon monoxide can bind to haemoglobin at the site where oxygen is normally bound.

● Oxygen transport can also be impaired due to the formation of methaemoglobin, i.e. oxidized haemoglobin, with the iron existing in the ferric instead of the ferrous state. Methaemoglobin is incapable of binding reversibly with oxygen.

Different toxicants can enhance the formation of methaemoglobin:

 nitrite;

 aromatic amines;

 some aminophenols;

 arylnitro compounds; and

 N-hydroxyarylamines.

Infants younger than 4 months of age who are fed formula diluted with water from rural domestic wells are especially prone to developing health effects from nitrite or nitrate exposure. The high pH of the infant gastrointestinal system favours the growth of nitrate-reducing bacteria, particularly in the stomach and especially after ingestion of con-taminated water. The stomach of adults is typically too acidic to allow for significant bacterial growth and the resulting conversion of nitrate to nitrite.

A proportion of the haemoglobin in young infants is still in the form of fetal haemoglobin.

Fetal haemoglobin is more readily oxidized to methaemoglobin by nitrites than is adult haemo-globin. Therefore, infants, and especially prema-ture infants, are particularly susceptible. In addition, NADH-dependent methaemoglobin reductase, the enzyme responsible for reduction of induced met-haemoglobin back to normal met-haemoglobin, has only about half the activity in infants as in adults.

STRUCTURAL PROTEINS. The replacement of sulfur by selenium upon a chronic intake of high doses

of selenium may lead to altered structure and function of cellular components. Especially sus-ceptible are the cells that form keratin (keratino-cytes) and the sulfur-containing keratin molecule.

Selenium therefore weakens the hooves of animals and hair, which tend to fracture when subjected to mechanical stress.

Selenium in the form of SeMet is easily absorbed by the same mechanism as methionine. Inorganic selenium absorption seems not to be regulated and is quite high (>50%). Bodily storage of inor-ganic selenium from selenite or selenate occurs as the selenoamino acids SeCys and SeMet. It has been reported that selenium and/or selenoamino acids can be incorporated into sulfur-requiring sites during protein production and thus change the integrity of the protein structure. Cows sup-plemented with 50 mg of injectable selenium suf-fered severe claw problems in the postpartum period. It is very likely that the excessive selenium supplement was incorporated into keratin fibres of the maturing keratinocytes with the key Cys and Met sites replaced by SeCys or SeMet.

Therefore, critical disulfide bridge formation was reduced or inhibited during the cornification process, creating inferior hoof horn lacking struc-tural rigidity with poor integrity.

Lipids/membranes (permeability/fluidity) Lipids and lipophilic substances of different types are integrated parts of the cell membrane. Cell membranes are the target of a number of very different toxic agents. These include the phenom-enon where anaesthetic ether and halothane as well as other organic solvents accumulate in the cell membrane, thereby altering the membrane characteristics and among others the transport of oxygen and glucose into the cell. Among the met-als, lead can increase the fragility of the erythro-cyte membrane. The huge group of natural plant glycosides called saponins (see Chapter 6) may also interfere with the structure of cell mem-branes. A broader definition of the saponins includes the steroidal alkaloid glycosides found in potatoes. Usually, these glycosides have low oral bioavailability and toxicity, but when given intravenously many show strong toxicity and cause haemolysis (rupture of the erythrocyte membrane). The general membrane-disrupting properties of saponins include pore formation by an unknown mechanism.

Nucleic acids

Covalent binding between a toxicant and a base in a DNA string (alkylation) can result in the forma-tion of a point mutaforma-tion (base substituforma-tion or insertion/deletion) upon replication of the DNA. If not repaired such a mutation may further lead to cell death or to the development of cancer. Alkylating (mutagenic) substances in food include two differ-ent types, among others, both of which must be activated through metabolism in the liver (and else-where) to become the actual mutagens. These are the mycotoxins called aflatoxins and the so-called frying mutagens within the group of PAHs, the most potent of the latter being benzo[a]pyrene.

10.2 Dose–Response Relationship Toxic effects may as we have seen be due to interac-tion with different targets through binding or chemical interaction. Regardless of how an effect occurs, the concentration of the toxicant at the site of action controls the effect. Dose–response data are typically plotted with the dose or dose function (e.g. log₁₀ dose) on the x-axis and the measured effect (response) on the y-axis. Because a toxic effect is a function of dose and time, such a graph depicts the dose–response relationship independent of time. Measured effects are most often recorded as maxima at the time of peak effect. Effects may be quantified at the level of molecule, cell, tissue, organ, organ system or organism.

An effect (response) may be quantal or graded.

An example of a quantal response is the percentage of dead animals in an experiment to describe the acute toxicity by determining the so-called LD₅₀ value (see Chapter 13), while an example of a graded response could be the increase in blood pressure for a single individual depending on expo-sure dose or the average increase in blood presexpo-sure for different groups of animals depending on the dosing of the group in question. Graded responses can be transformed to quantal responses. Looking at the example with blood pressure this could be done by making a graph of the percentage (the effect/response) of animals that experience at least a 10% increase in blood pressure as a function of the dose. Let us now look at such curves and get to know the possible differences. Two hypothetical dose–response curves are shown in Fig. 10.2.

Both the shape and slope of the dose–response curve are important in predicting the toxicity of

a substance at specific dose levels. Huge differences among toxicants may exist not only in the point at which the threshold for the toxic effect is reached, but also in the response increase per unit change in dose (i.e. the slope). In the example shown in Fig. 10.2, toxicant A has a higher threshold but a steeper slope than toxicant B.

A given toxicant will normally have different adverse effects, each of which may start at a differ-ent threshold. An example could be the differdiffer-ent effects of intoxication with methylmercury.

Figure 10.3 shows the frequency (% affected) of different symptoms (y-axis) seen for adult victims of intoxication with methylmercury in Iraq (1987)

Threshold

Toxicant A

Toxicant B 100

50

Response (%)

10 0

b

Increasing dose

b a

a

Slope = a b

Fig. 10.2. Hypothetical dose–response curves for two different toxicants, A and B. Adapted from The Encyclopedia of Earth web page.¹

0 10 20 30 40 50 60 70

Frequency (% affected)

80

0 200 400

Hair Hg concentration (ppm)

600 800

Paresthesia Ataxia Dysarthria

Deafness Death

Fig. 10.3. The dose–response relationship for different adverse effects of methylmercury intoxication (frequency of response versus hair concentration).

due to the use of mercury fungicide-treated cereals for human consumption (bread making). The dose (x-axis) is in ppm of mercury (Hg) as measured in the hair (a biomarker for the exposure).

10.3 Conclusion

Toxic effects are the results of interactions between a toxicant and one or more target molecules in one or more target tissues or organs. Any toxic constitu-ent will normally show a number of differconstitu-ent toxic (adverse) effects, each of which will be characterized by a threshold concentration/dose beyond which the effect may be seen. A dose–response relationship will exist, which can be described by a graph. The graphs for different effects of the same compound, as well as for toxic effects of different compounds, may exhibit different slopes, i.e. the response increase per unit change in dose may vary.

Note

1 The Encyclopedia of Earth (2008) Dose–response relationship. Yuill, T. and Miller, M. (eds). EIC Secretariat,

National Council for Science and the Environment, Washington, DC; available at http://www.eoearth.org/

article/Dose-response_relationship

Further Reading

Heinrich-Hirsch, B., Madle, S., Oberemm, A. and Gundert-Remy, U. (2001) The use of toxicodynam-ics in risk assessment. Toxicology Letters 120, 131–141.

Klaassen, C.D. (2008) Casarett & Doull’s Toxicology: The Basic Science of Poison, 7th edn. McGraw-Hill, New York, New York.

Lu, F.C. and Kacew, S. (2009) Lu’s Basic Toxicology:

Fundamentals, Target Organs, and Risk Assessment, 5th edn. Informa Healthcare, New York, New York.

Niesink, R.J.M., de Vries, J. and Hollinger, M.A. (1996) Toxicology: Principles and Applications. CRC Press, Boca Raton, Florida.

Omaye, S.T. (2004) Food and Nutritional Toxicology.

CRC Press, Boca Raton, Florida.

Tozer, T.N. and Rowland, M. (2006) Introduction to Phar-macokinetics and Pharmacodynamics – The Quanti-tative Basis of Drug Therapy. Lippincott Williams &

Wilkins, Philadelphia, Pennsylvania.

11.1 The Beginning – Food Regulations History (the USA with FDA, USDA, etc.) The modern history of food regulations with respect to chemical substances potentially found in food-stuffs starts in the USA with the 1958 Food Additives Amendment to the Food, Drug and Cosmetic Act of 1938. The amendment regulated the use of food additives such as salt, nitrate, organic acids and other chemical compounds. The Food Additives Amendment requires FDA approval for the use of an additive prior to its inclusion in food. It also requires the manufacturer to prove an additive’s safety for the ways it will be used.

In writing this piece of legislation the American Congress felt, however, that a number of substances intentionally added to food would not require a formal scientific premarket review by FDA to assure their safety and hence approve their use.

Such trust in a compound could, for example, be based on its safety being ‘established’ by a long his-tory of use in food. Thus, a two-step definition of a ‘food additive’ was adopted in the Amendment.

The first step broadly included any substance, the intended use of which results in it becoming a component or otherwise affecting the characteris-tics of food. The second step, however, excluded from this definition substances that are:

Generally recognized, among experts qualified by scien-tific training and experience to evaluate their safety (‘qualified experts’), as having already been adequately shown through scientific procedures (or, in the case of a substance used in food prior to January 1, 1958, through

either scientific procedures or through experience based on common use in food) to be safe under the conditions of their intended use.

Salt, sugar, spices, vitamins and monosodium glutamate were classified as GRAS substances, along with several other substances.

FDA therefore published a list of these generally recognized as safe (GRAS) substances on 9 December 1958. This list came to be called the GRAS list. Following the publication of the GRAS list, FDA made no systematic attempt to evaluate available scientific information on the GRAS sub-stances. By 1969, as a result of a recommendation in the report of the White House Conference on Food, Nutrition, and Health, President Richard M. Nixon directed FDA to make such a critical evaluation of the safety of GRAS food substances.

The following GRAS review for years became a major project at FDA’s Bureau of Foods.

In 1960, the US Congress passed similar legislation governing colour additives. The Color Additives Amendment requires dyes used in foods, drugs and cosmetics to be approved by FDA prior to their mar-keting. In contrast to food additives, colours in use before the legislation were allowed continued use only if they underwent further testing to confirm their safety. Of the original 200 provisionally listed colour additives, 90 have been listed as safe. The remainder have been either removed from use by FDA or withdrawn by industry. Both the Food Additives Amendment and the Color Additives Amendment prohibit the approval of an additive if it is found to