Mammalian Toxicology, Session 2

Toxicant targets; Physiologic dose-response; C 2, 21; The role of intercellular chemical communication: hormone, receptor, transducer, effector; agonist, antagonist; Interconnections of transduction mechanisms; C 3; End of Add/Drop

Toxicant targets:

Forms of lethality and dosages:

Before going into any detail with respect to the targets of toxins or toxicants we need to repeat a few words that were mentioned in the in-house session last week.  When we speak about toxicity what do we really mean?  What about toxicity?  As a toxin can have a lethal or a sublethal effect, is one word adequate to define it?  It would seem there is a need to define varying types and levels of toxicity or toxic response.

Lethality: mortality, death (induced reasonably rapidly as an obvious consequence of exposure)

Morbidity: a decrease in functionality that may ultimately lead to a decrease in lifespan

Reduced reproductive capacity: decrease in reproductive output (this has a genetic carry-over to the next generation because there is a resulting change in the potential gene pool)

Decrease in specific functions or acuities (sensory: sight, hearing, taste, smell, touch; specific metabolic functions - food allergies; appearance - hair growth, acne): these decrease quality of life but are often nonlethal, do not decrease lifespan and do not alter the gene

Reduced asthetics or other quality of life issues: these are often issues that have little or no medical impacts except at the psychological or emotional levels

Reduced quality of the environment: this may impact any or all of the above items

Note that these range from severe immediate impacts on the individual, through prolonged or delayed impacts on the individual or the species, to subtle effects that may only alter survival or functions of individuals or species via alterations of behavior or mental vitality.   Evaluating risks of toxic exposure then may encompass analysis of biological risks and exposure tests, potential medical or economic risks of exposure to or lack of exposure to the toxicant of interest, and possible impacts on the integrity or improvement of environmental quality.  The overall picture may involve a variety of competing interests or benefits and definitely involves a cost/benefit analysis.  Even at the individual level this is reflected in the statements that exist on drug and household chemical packaging.  For medications there are not only directions for proper use, but indications of possible deleterious side effects.  These are often unrelated to the drug target tissue of interest, e.g., antihistamines not only decrease swelling in nasal mucus membranes, they often suppress mental alertness and/or increase blood pressure.  The detailed analysis of positive and negative drug impacts is subsumed in the science of pharmacology but has obvious overlap and relation to toxicology both in its tenets and methodologies but also in the subjects of study and the analyses used to evaluate study results.

Targets of toxic insult:

A chemical can act on an organism in two ways: directly and indirectly.

To act indirectly, it may disrupt the functioning of a cell or tissue that communicates with, regulates, or supports another cell or tissue.  Thus, a disruptor of neural or pituitary control over adrenal function may generate a toxic response that is due to failure to produce proper amounts of the glucocortical steroids that are a main product of the adrenal cortex.  So although the toxicant is acting in tissues in the head its manifested toxic response may appear in altered immune function (suppressed by high glucocorticoids) or in impaired growth (which requires the anabolic actions of glucocorticoids) of body structures.  Obviously, teasing apart such indirect actions requires a keen knowledge of the normal regulatory circuitry within the body.  The importance of background knowledge of endocrinology, neurobiology, and immunology to a toxicologist cannot be overstated.

To act directly, it usually has an impact on the cells and tissues that can be either general or specific.  If it acts generally, it does so via a physical or chemical reaction that can be explained in generic terms: corrosivity - high acidity, highly caustic; chaotropism; detergency; solvency; chemically reactivity - forming adducts with broad classes of compounds like amines, alcohols, aldehydes, ketones, or alkenes that are present in many biological macromolecules; or generally proteolytic, lipolytic, or glycolytic - for example enzymes that might be found in bacterial or mycotic toxins or snake venoms.  These actions can occur in nearly any or all exposed tissues or cells so the site of exposure will normally manifest the highest degree of toxic insult.  Other toxic responses may also be induced in a secondary, or indirect, manner such as sensitization of the immune system via antibody production against chemically altered proteins or DNA in the originally exposed tissue, compromise of hearing if the tympanic membrane is disrupted by chemical or toxic actions.

The role of intercellular chemical communication: hormone, receptor, transducer, effector; agonist, antagonist:

If a toxin or toxicant is to act with some specificity as many, many do, it must interact with the cellular and tissue target in a specific manner.  To do this it has to mimic or structurally resemble a molecule that is endogenous which binds to a particular location on or within a macromolecule in a cell or tissue.  This might be an enzyme reactive site, an ion channel within a cell membrane, the receptor protein for a particular hormone in the membrane or nucleus of a cell, or a particular site on a DNA molecule.  Note that all these locations have evolved shapes that allow them to act as conduits for receiving, transducing, or acting on chemical information passed to them from outside the cell or from other parts of the cell.  That they have evolved these shapes suggests that they have done so in response to "normal" inputs, not extraordinary ones that may have been developed by synthetic chemists within the past 50 years.  This means that such chemicals are indeed acting as structural mimics to endogenous or frequently encountered environmental chemical cues (see receptor antagonists, C&D p. 17).  Moreover, there are finite numbers of these interaction or binding sites within cells or tissues.  Thus, such interactions are saturable at high enough concentrations of the toxicant involved. Also, because there are finite numbers of these sites, toxin or toxicant interactions are normally in competition with the binding of endogenous chemicals (ligands).  The relative strengths of the interactions of the binding site with the endogenous ligand and with the toxicant (the affinity constants for binding endogenous ligand or toxicant) will help determine the effectiveness of the toxicant in acting on the tissue.  Along with the actual concentration of the binding site and the concentrations of the endogenous ligand and the toxicant, the saturation of the binding site by these two compounds can be computed based on simple binding kinetics: [BT] == [B][T]Kbt, [BE] == [B][E]Kbe, and BT <==> T + B + E <==> BE, where B is the Binding site, T is the toxicant, and E is the Endogenous molecule.  Note that concentrations of B are often on the order of picomolar to micromolar as are the concentrations of E, that the dimensions of Kbe are often in the range of 108 to 1011 M-1 (=L/M), and that the resulting saturation for B sites is often below 50 or even 10%.

While this may describe the biophysics of the binding of a toxicant, the biochemical actions may range from strict competition with the endogenous ligand to allosteric (other site) modulation of the actions of the endogenous ligand via binding to locations on the target, binding, molecule that differ from the endogenous ligand (though they may overlap sterically).  Toxicants that are termed "agonists" will not only mimic binding to a site overlapping that of the endogenous ligand, but they will trigger a similar cascade of intracellular events that is identical to, or strongly similar to that of the endogenous ligand.  This usually occurs because they alter the shape of the B molecule in a way similar to E and cause it to allosterically interact with other molecules, transducers, that act to transduce the initial interaction into intracellular signals that the cell then converts via other macromolecules, usually protein effectors, into metabolic and/or physical reactions.  On the other hand, toxicants, that simply block the E binding site but do not initiate the same sequence of events that E does, are termed "competitive antagonists."  Agents that block the actions of E without competing directly for its binding site would be "noncompetitive antagonists" or "uncompetitive antagonists" depending on the physicochemical description of the kinetics of the interaction.  These kinds of antagonists may mimic the product or transition state(s) of an enzyme or they may cause formation of a nonproductive form of enzyme complex, receptor-ligand complex, or DNA-ligand complex without necessarily blocking the binding of the endogenous ligand. In endocrinology and neurobiology we also encounter molecules that bind to receptors which trigger cascades of signals within cells that modulate or counter the actions of endogenous ligands acting via different receptors.  Clearly these are a form of noncompetitive antagonists (or agonists) that does not fit the frame of binding to a single target molecule. Toxins may well act in a similar manner (see also functional antagonists in C&D p. 17).

Interconnections of transduction mechanisms:

It is worth noting that many of the intracellular pathways linking extracellular chemical information inputs to cellular action have been defined (cf. any biochemistry, physiology, or endocrinology text).  Moreover, as these have been more closely examined, it has also become clear that there are many instances in which these intracellular cascades cross and interact.  That means that toxins may act via one or more of these pathways to activate or inhibit their normal functions and/or to change they way in which they respond to the usual extracellular signals. The following provide a couple of illustrations of this point:



If toxicants act indirectly, in an antagonistic manner by means of speeding the clearance of an endogenous molecule like a steroid or another toxicant via action at a site other than a receptor or target enzyme, they would be termed a dispositional antagonist.  If they compete for metabolism they might well act as synergists, increasing or potentiating the impact of the endogenous ligand or another toxicant.

In a few cases a toxicant acts not on a receptor but on a ligand or another toxicant directly, similar to the interaction between an antigen and an antibody.  In these cases the first toxicant will be a chemical antagonist of the ligand or second toxicant.

Targets can also be general or specific.  The generalized targets fit with nonspecific toxic insults as mentioned earlier: burns, sensitizations, tissue trauma resulting in necrosis (cell death normally induced by osmotic disruption of cell membranes caused by drastic alterations of cellular environment, low or high pH, solvent or detergent disruption of membranes, etc.).

Specific targets are often associated with specific organs, tissues, or cells.  These in turn reflect special features of those organs, tissues, or cells often translating into similarities in surface proteins, lipids, or carbohydrates, or particular receptors or ion channels.  Such entities will concentrate available toxicant molecules that bind to such molecular targets and will cause those tissues to act either as sites of toxic insult or as sites of toxicant metabolism, biotransformation and clearance.  Note that sites of accumulation, for example fat depots accumulating lipophilic compounds via nonspecific physicochemical interactions between the toxicant and stored lipid molecules, will also concentrate toxicants.  But they often lack target molecules of sufficiently high avidity for the toxicant molecules to bind them preferentially to those targets rather than the nonspecific lipid stores.  When toxicants have no specific molecular targets, they often fail to elicit a specific toxic response.

Specific binding of toxicants also often reflects the status of another equilibrium in plasma and/or extracellular fluid, that is, the carrier or circulating binding protein bound form of the toxicant versus that found free in solution.  Most lipophilic compounds and many other small ionic compounds tend to bind to sites on circulating carrier proteins which the body normally employs to distribute endogenous hormones and other molecules to their target tissues.  Many of these carrier proteins are synthesized in the liver and often have binding affinity constants, Ka's, that are 10 to 1000 times lower than those of specific cellular receptors for a given hormone or endogenous molecule, i.e., 106 to 109 L/M.  This allows such proteins to increase the volume to which endogenous ligands are distributed prior to loss of the ligand to metabolic, clearance, or simply receptor binding sites.  They buffer the concentration of ligand in circulation and extend its biological "half-life" by decreasing its clearance from circulation.  But these carriers often accept molecules of similar size or shape to their natural ligands.  So toxicants can often be carried and distributed in this manner.  Such carriers are rarely saturated with natural ligand beyond 50% of capacity, so there are often ample "spare" sites for toxicants to be carried even without interfering with natural ligand capacity.  The binding reactions to such carriers are noncovalent and reversible.  As free ligand increases in plasma due to endogenous synthesis or exogenous exposure, binding increases: B + L <==> BL, and the reaction proceeds to the right.  As the complexes move to sites of lower free ligand, L, concentration, the reaction proceeds to the left generating more free ligand that can then bind to sites of higher affinity such as hormone receptors or specific toxicant binding sites.  If free ligand is cleared by metabolic processes, the reaction also proceeds to the left decreasing the amount of complex remaining.  Obviously, if enough carrier protein exists to tie up the majority of a toxicant until it is cleared, the biologically available concentration of the free toxicant many never exceed the minimum needed to generate a toxic insult.  However, if metabolic or genetic alterations greatly diminish carrier binding capacity, a toxicant dose that may be readily tolerated by a normal individual may severely compromise the metabolically or genetically altered individual.  Finally, note that many ligands can bind to several types of carrier proteins. Albumin acts as a remarkable "sink" for many lipophilic and ionic compounds but subclasses exist for many hormones, vitamins, and lipids (cf. C&D, p. 119-121.)

Selective Toxicity:

There are differing forms of toxicity.  Acute lethality, effects on specific organs (that may or may not be potentially lethal), chronic, or subchronic effects (again that may or may not be ultimately lethal).  In the case of chronic responses, there is historically a considerable emphasis on carcinogenesis as the endpoint of most concern.  But what about neural degeneration, arthritic conditions, vascular rigidity, loss of muscle mass, loss of bone mass, or decline in immune function?

Note the definitions of lowest observable adverse effect level, LOAEL, and no observable adverse effect level, NOAEL. Note their dependencies on abilities to measure the chemical and the response within the biological system.

Physiologic Dose-Response:

While we looked at a few of the characteristics of the dose-response curve last time, there are more items to cover.

Definition of a toxicant and assumptions in dose response:

The empirical definition of a toxicant can be constructively viewed as a parallel phrasing of Koch's 1890 postulates regarding the empirical definition of a pathogen ( http://alan.kennedy.name/crohns/primer/koch.htm http://www.sci.wsu.edu/bio/micro310koch.html )

  1. The specific organism should be shown to be present in all cases of animals suffering from a specific disease but should not be found in healthy animals.
  2. The specific microorganism should be isolated from the diseased animal and grown in pure culture on artificial laboratory media.
  3. This freshly isolated microorganism, when inoculated into a healthy laboratory animal, should cause the same disease seen in the original animal.
  4. The microorganism should be re-isolated in pure culture from the experimental infection.
Note that virtually the same empirical definition can be applied to hormones by substitution of "hormone" for "specific organism," by substituting "normality" for "suffering from a specific disease," by substituting "chemical or hormone" for "specific microorganism," and by substituting "purified to homogeneity" for "grown in pure culture."  The idea is that a hormone is present and can be isolated from a normal animal and given to a deficient one to correct the deficiency.

In the case of a toxicant, it will be absent from a normal animal but can be isolated in pure form from an intoxicated animal.  The toxin can then be given to a normal animal and demonstrated to generate the same symptoms of intoxication so long as it is present (in sufficient amounts).

 Casarett and Doull (p. 22-23) indicate there are three assumptions underlying dose-response relationships:
  1. The response is due to the chemical administered.
  2. The magnitude of the response is in fact related to the dose [because]
  1. There exists both a quantifiable method of measuring and a precise mans of expressing the toxicity.
Note how this depends on the existence of molecular response machinery virtually identical to that needed for a hormonal response.  Also, that it demands that we have assays and chemical measurement techniques that are capable of identifying both the chemical in the responding system and the physiological response of the physiological system to the chemical.  In endocrinology we spend a good deal of time talking about the distinction between hormonal mass and hormonal potency because we can measure mass quite apart from a biological response system.  But since hormones are messenger molecules with no other task in the body, we cannot identify their function and biological activity (potency) unless we place them into an appropriately responsive system [and unless we have a sufficiently specific and sensitive means to observe the response of that system].  The same is true of toxins and toxicants.  These chemicals can be measured physically, but such measurements are of limited utility unless we can relate them to the actions of the chemical on a physiological target system.

As in endocrinology, the action of a chemical in a physiological system depends on a large variety of parameters.  Among the more important are the actual biochemical state of the molecules in the biological system.  Are they of the correct stereoisomeric form to be functional?  If macromolecular, are they denatured?  Are they being sequestered from tissues by binding proteins, fat depots, particulates in the gut?  Is there an appropriate receptor present in a target tissue?  Are there enough of those receptors to allow a meaningful response to the chemical?  Are protective mechanisms present that can degrade or remove the chemical before it can act on the target?  Where is the dose being delivered relative to the responsive target and the existing protective mechanisms?

Potency vs Efficacy:

These are shape characteristics of the dose-response curves and are dictated by the kinetics of binding of toxicant (or hormone or drug) to the receptors [or target molecules] of the target tissues.  

Potency refers to the range of doses over which a chemical acts; A is more potent than B [in the same response system] if the range at which A exerts its effects is lower (less A required) than the range required for B.  This may reflect a higher Ka for binding of A than B or the use of a somewhat different receptor for A that is present in higher concentrations than that for B.  It could also indicate the absence of clearance or interfering systems for B rather than A.

Efficacy refers to the upper limit of response of the biological system to a chemical.  A and B may be equally effective even if A is more potent than B, but C and D may be equally potent, generating responses in the same range, while C is less efficacious because the maximal response to C is less than that seen with D.  Differences in efficacy probably result from differences in the receptor(s) being used by the two toxicants (or hormones or drugs) or from the presence of protective or clearance systems that limit the impacts of one toxicant versus another.  It may also result from differences in the post-binding cascades that ultimately lead to response.  It will not result solely from Ka differences in binding the same receptor, though it may result from differing fits in the same receptor where one toxin fully activates a post-binding cascade while another only partially activates the same cascade (and may activate others instead, including others that interfere with the one being observed).

Looking at the dose response curves often presented, note the use of probits for the Y, response, axis.  The use of raw dose versus raw response would usually yield a curve that looked hyperbolic.  Plotting this as log dose versus raw response generates a more classical sigmoid curve that actually depicts the cumulative probability of observing a response to a given dosage of molecules.  Plotting log dose versus log response often linearizes the response curve significantly, but to fully straighten it the y-axis must be recast as a probability function.  The probability function normally used is the probit which anticipates the first derivative (change in response over change in dose, dR/dD) of the curve that will depict a Gaussian, normal curve.  Using this assumption the axis is plotted as multiples of the standard deviation of the mean of that curve above and below the mean (= response value at 50% of maximal response).  Note that use of a second derivative curve (d2R/dD) would provide a sharp deflection at the point of 50% response and might be useful in some forms of dose comparisons across compounds or similar systems.

A bit of additional discussion of probits and logits may be found at: http://www2.chass.ncsu.edu/garson/pa765/logit.htm
Possible Case Studies:
  1. DDT and persistent pesticide bans
  2. Dredging of the upper Hudson for PCBs 
  3. Zero tolerance for Alar in apples
  4. Labeling and restriction of milk from cows injected with or transgenic for bovine GH
  5. Oral contraceptive contamination of municipal sewage effluents in Great Britain
  6. Plasticizers such as phthalates as environmental anti-androgens or generating estrogenic effects in dialysis patients.
  7. Bacillus thuringenesis (BT) toxin cloning into plants and environmental dispersal.
These should take into account the following items:
  1. Mammalian associations
  2. How does toxicology play a role in these cases?
  3. What are the assumptions, stated or unstated, that support the contrasting arguments in these debates?
  4. Are risk assessments being used?  If so, are these realistic and well grounded
  5. What particular elements of toxicity testing and modeling are being employed?  Give examples.
What about Hanford radiation ground water contamination case?  Single cases such as this may have a relatively straight-forward explanation and resolution.  In such instance, with respect to the projects for this class, contrast this with other similar risk assessment cases, e.g., storage site contaminations or studies on possible contamination in South Carolina or the new site in Nevada.  Alternatively, look at contaminations at Three Mile Island, PA versus Chernobyl, USSR perhaps focusing on only a single isotope like 131I.  The intent of contrasts for environmental cases being to see if the assessment strategies were similar and if the public and private responses to the cases were justified and/or adequate.  Other examples might be the groundwater contaminations in Woburn, MA, those on Cape Cod near Otis Air Force Base, those at Love Canal, NY, or those associated with the tanning industries or gasoline storage.  Sources to include in case studies would be books, journal articles, websites, newspaper articles, regulatory documents or laws, maps, and images resulting from the cases.
What about food safety history?  Upton Sinclair's The Jungle which depicted conditions of workers in the Chicago meat packing industry in the early 1900's initiated a public response that resulted in Congressional action and the passage of the earliest pure food legislation.  This eventuated in the creation of the Food and Drug Administration and became the model for other regulatory agencies charged with limiting public risks.  Examination of recent cases or present day conditions in the poultry industry, meat packing, and fish processing would seem very suitable as case studies as would an historical approach to development of any of several of the existing agencies charged with risk assessment, limitation, and reduction. New opportunities to look at food safety might also involve the handling and management of transgenic organisms such as grains or animal products.  Do genetically modified foods/crops require FDA approval, for example?  A German standpoint is given in: http://www.gtz.de/biotech/dokumente/biotech2.pdf and a relevant paper on Bt toxin safety is given in http://web-mcb.agr.ehime-u.ac.jp/gmo1/english/Bt%20Crops-Advanages.pdf .  The actual registration document for Bt modified plants is at http://www.agbios.com/articles/2000264-A.pdf .

Introduction to risk assessment:

The business of much of toxicology is the evaluation and establishment of the type and degree of risks posed by exposures to particular toxicants or toxins.  Risk assessment is a set of procedures intended to look at the cost/benefit ratio of exposure versus non-exposure, or exposure versus lesser exposure. It involves a detailed examination and evaluation of alternatives to exposures and the various potential costs involved in exposure versus those alternatives.  The alternatives are not necessarily simple substitutions, they may be entirely different solutions to a given technical or social problem, they are often "apples and oranges" such as economic benefit versus medical or ecological risk.  Such evaluations are often decided via a set of contrasting counter arguments with the most logical or powerful debater in a given dispute prevailing over those with less strong arguments or information. As such, these debates often have no "right" or "wrong" answer.  To explore the issues involved it is best to examine examples of scenarios or case studies.  Several examples of risk assessment debates affecting public health include: the AIDS epidemic as viewed from the early 1980's (see The Band Played On), the debates concerning DDT and persistent chlorocarbon pesticides in the late 1960's and 1970's (see Silent Spring and many others), use of tetraethyl lead in gasoline debated in the 1970's, use of diethylstilbesterol as a lifestock fattening agent debated in the 1970's and 1980's.

The kinds of testing needed to bring a product to market (even when not intended for human consumption) is outlined in the following site on agricultural products: http://www.ento.vt.edu/~mullins/pestus2001/notes/lecture/Lec24.html

Additional descriptions of the risk assessment process and strategies may be found at: http://www.riskassess.org/

Discussion Questions for Session 2

Are the questions being asked in the distributed sets too broad?  Possibly, however, the intent is to force class members to not only review the text material on a given topic, but to integrate information on other related issues or topics and/or to analyze the information or situation presented or referenced.   Although Casarett and Doull is a great text, it suffers from the channeled thoughts of an established discipline that sometimes fails to understand or incorporate information or advances that have taken place in related disciplines or in areas that directly impact toxicology, e.g., developments in genetic engineering or advances in endocrinology.  The questions are attempts to push beyond the banks of the channeled stream.

Chronic Toxicity & Carcinogenicity (QS1Q6)
  1. Note the emphasis of chronic toxicity testing and mutagenesis testing on carcinogenesis.  Is this emphasis currently appropriate, or are there other possible chronic outcomes to be concerned with?
Toxin Metabolism Considerations (QS1Q7)
  1. Emphasis in the discussion of biochemical and physiological means of toxin metabolism and clearance is on the adult animal. Are there other important considerations or routes to be considered in growing offspring or during the reproductive process?
Oral Toxicity Paradox (QS2Q6)
  1. Oral toxicity studies have defined a paradox of this route of exposure. What is the paradox and how might it be explained? A somewhat similar paradox can arise if dietary insoluble fiber is high and exposure is again oral. Why might this occur? Are either of these situations of possible use in treating acute poisonings? In chronic intoxications?