Mammalian Toxicology, Session 12

Discussion of Draft Projects & Discussion Questions

It is probably wisest to read the material listed under Session 13 prior to next week.  That material will be relevant to the new Discussion Questions listed and needs to be digested prior to the Final.

Final Exam

Next week, May 4, I will post a set of 10 point questions in conjunction with the lecture posting.  These constitute the first half of the final exam. You will be responsible for choosing 5 of these to answer and submit before May 11.  On May 11 you will answer a series of 10, 5 point, questions that constitute the other half of the final. All responses will be in class or via the Prometheus, on-line, site.

Discussion of Draft Projects

Please note that I made the following notations independent of comments made by students.  They may therefore be redundant or contradictory to other critiques.  Please use them in conjunction with any other available commentaries as well as your own judgments to revise and improve the penultimate projects to make them suited to final submission and posting to the course Web site.

Group 1: ????

Group 2: ????

Discussion Questions

Agencies & Risk

3. Modern toxicology includes the concepts of causality, risk assessment, and site specificity of action.  Why are all these concepts relevant to the following agencies: USPHS, NSC, EPA, FDA, OSHA, NRC?  How many of these agencies derive directly from concerns about toxicant exposures?

Most of you responded by looking up each of these agencies and noting their mission statements, all of which relate to public health and safety.  The National Institute of Environmental Health Sciences as a part of NIH within USPHS is notable in having a very high profile with respect to toxicology and toxicological studies.  If you missed that one, check it out.  Also look at the historical roots of the USPHS and the FDA.  They are a lot longer than you might imagine but they did arise directly from concerns about toxicant exposures.  EPA arose from the Federal Water Quality Agency (also known as the Federal Water Pollution Control Agency) in the early 1970's both of these entities were responses to the likes of Rachel Carson.  I worked as an analytical chemist evaluating PCBs for FWQA briefly in 1970; my dissertation work was supported in part by EPA.

Food Safety

11. What steps are required for drug product clearance by the FDA?  How are food supplements handled by the same agency? Are there additional or alternative rules for genetically engineered foods or drugs produced by genetically engineered organisms? Do such rules seem adequate to assure safety of humans using genetically engineered organisms or products?  What about environmental safety of these organisms or products?

The steps of testing leading up to clinical trials are enumerated by several people as are the lack of such steps for food supplements and genetically engineered foods.  Note, however, that genetically engineered pharmaceuticals do go through that same series of tests.  Food toxicology contains a lot of contradictions, twists and turns because of the ubiquity of chemicals and their essentiality for the maintenance of life.  Organisms cannot live without food and they cannot eat without taking in and absorbing compounds that have undesired effects.  The idea of regulators is to minimize, not eliminate, exposures to deleterious compounds for the majority of the susceptible population.  That's obviously tricky, especially when legislation like the Delaney clause exists (you need to know what that is, by the way).  Do note the way food contents are classified and subclassified for regulatory purposes and that some subclasses are regulated or require testing while others do not.

With respect to genetically engineered foods, most of you felt they were not adequately regulated.  How do you feel about new poultry breeds or bean hybrids derived by traditional breeding methods?  Note that many of these will contain mutations, proteins, or secondary metabolites that do not exist in the parent strains.  Should meat or seeds from these also be put through thorough toxicological testing?  In other words, where do we draw the line for what requires testing and what does not?

NOAEL, LOAEL & Threshold Model

12. Are the NOAEL and LOAEL concepts most compatible with a threshold level or a no threshold level version of dose responses?  How do they differ from zero dose?  Does the last answer change for environmental exposures if newer methods allow lower limits of detection for the toxicant in question?

A zero dose, is a zero dose, that is, no toxicant given.  That may not be the same as absolutely no exposure if the toxicant is ubiquitous.  Under those circumstances the zero dose would be that tested group that had been given no measurable toxicant above that found in the environment.  In all tested groups there is variability of response as measured as a parameter on the y-axis of a dose response curve.  That variability means that there will always be some uncertainty about the response at zero dose and there will always theoretically be a range of nonzero doses that cannot be distinguished by bioassay from zero because each of their ranges of response will overlap that seen for the zero dose.  The first dose that lies statistically beyond that range of uncertainty is the LOAEL.  The last dose that lies within that range is the NOAEL.  If enough doses are tested with enough animals there will always be a region of low doses that falls below the LOAEL and the data can be described by a threshold model.  If very few doses are tested, it will be difficult to see this region and the data would almost automatically need to be fitted to the no threshold model.

Note that what becomes tricky is not this conceptual formulation of the problem but its practical application.  I can dilute a dose serially with a great deal of confidence before administering it to animals.  But in testing, what is important is the biologically available dose.  And to know what that is requires an independent assessment of the levels of the toxicant in the test animals.  This is where the issue of analytical sensitivity comes in.  If I cannot measure something in the animal, then the response should be grouped with the zero dose group.  And it may well be that my measurement methods do not allow me to "see" the toxicant in the test organism until I reach beyond the statistical line separating NOAEL and LOAEL.  In that case, the data will only fit a no threshold model.

Repair Functions & Toxicity

14. How do repair functions complicate the analysis of toxicity in acute exposure models?  What about chronic exposure models? Do they have the same impact on results in adults as opposed to developing animal models?

Repair functions take time so they are most important for chronic models.  But "acute" in toxicology also does not mean only a few minutes.  It may well be that some repair functions may take place within the timeframe of even acute tests so that they cause an increase in the NOAEL or threshold for toxicity.  They would have similar impacts on chronic models, and, indeed, may make some toxicants appear benign in such systems.

As to which systems would be most sensitive to toxicant insult, the general rule is that developing, aging, and immunocompromised individuals will demonstrate the least effective repair functions and therefore be susceptible to the lowest doses of toxicants.  Obviously there will be exceptions, especially where, for instance the ultimate target of a particular toxicant does not yet exist (or no longer exists) at the stage of toxicant exposure or where physiological mechanisms, e.g., fetal hemoglobin affinity for oxygen, allows functioning in the presence of toxicant that would not normally occur in the young adult/adult.

Toxicology Information

15. Search out the following sites and explore them: HazDat, EXTOXNET, RTECS, Toxline, IRIS, IARC. What information do they contain?  Do they appear up to date?  Print out some examples of the contents and see if you can interpret them.  To what area(s) of toxicology are each of them relevant?

In former years many of the answers did a nice job of looking these up and actually exploring their contents.  Others did not tell us whether the databases were up to date, nor did they tell us anything about examination of the contents of the databases.

Temporal Changes in Toxicity

18. Given an increased knowledge of the metabolic pathways utilized by mammals to process toxicants, how would you now design an experiment in which two neuroactive drugs were to be used repeatedly to test particular brain circuits?  Would any of this make any difference to your interpretation of results in which there was an apparent decline with time in the responsiveness to one of the drugs?  Does this knowledge make you view the use of multiple drugs in elderly patients any differently?

Neurotransmitters and drugs that impact their actions, like many hormones in the periphery, have a habit of altering their target tissues.  They often cause a decrease in tissue sensitivity to the same compound in the short term.  For milliseconds to seconds this is due to depolarization/ repolarization, the neural refractory period.  For minutes to hours this may involve receptor down regulation in which the transmitter-receptor complexes are internalized by the target cell and decoupled and/or degraded.  For minutes to days it may also involve receptor desensitization which is usually associated with alteration of the intracellular signaling pathways so as to make them unresponsive to a like stimulus until new conditions appear, or new signal proteins are synthesized.  Obesity related diabetes is a good model that demonstrates how continuous stimulation with a hormonal signal, insulin, leads to down regulation/desensitization of its own receptors, generating a refractive, unresponsive or insensitive state in insulin target tissues.  Opiate abuse produces a similar state in the CNS that leads addicts toward an upward spiral of dosage needed to attain a given state of intoxication.

There are also cases, e.g., estrogen sensitive tissues, in which a stimulus up-regulates it's own receptors making the target tissues more sensitive to a second dose of hormone/neurotransmitter or agonist.  Likewise a lack of regular stimulation, e.g., limb denervation, can also lead to a state of hypersensitivity marked by the presence of increased numbers of neuroreceptors at the cell surface.  Blockers of presynaptic release often generate hypersensitivity states in the post-synaptic neurons.

And there are also instances where treatment with one hormone or neurotransmitter up-regulates the response to a second hormone or neurotransmitter, e.g., progesterone primes endometrium to the actions of estrogen, estrogen primes gonadotropes to the actions of LHRH.

At the level of the organism, one drug can often modify the activation or deactivation of a second by means of altering the activities of phase I or II enzymes in the liver or the activities of enzymes such as the MDRs or OATs systems in the kidney.

So the acts of one neuroactive drug can impact a second neuroactive drug in a variety of ways that may be antagonistic, agonistic, competitive, additive, or synergistic.

How to test two such drugs?  Each would need to be tested by itself in a design that would include multiple doses administered at fixed intervals to each animal/subject.  Interaction groups would need to be used to explore the impacts of which drug preceded which and how any unique toxicities or actions of each drug were impacted by the other given simultaneously or before or after the other.  The affected brain circuits would obviously of most interest, but some attempts would be needed to explore hepatic functions (formation of metabolites in blood or urine) and the comparative absorption and concentration of the drugs in the brain areas of interest.

The elderly have declines in hepatic functions, and often suffer from compromised neural function.  Drugs may well have a prolonged half-life in circulation.  If the parent drug is active, this would prolong its actions.  If a metabolite is active, it might not reach effective concentrations until much later than in a younger individual, or, indeed, may not ever reach an effective level.  The elderly may not be able to tolerate a synergistic action of a second drug in the face of one acting on the same or interconnected neural tracts. Diana's comments they are particularly pertinent here.

DNA Size

19. Look up the dimensions for 1 base pair of DNA and calculate the length of 1 complete copy of cellular DNA (3 x 109 base pairs). Compute the volume of a typical cell nucleus, about 10 um (10 x 10-6 m) in diameter.  How often does the DNA have to be folded to fit in that volume?  Given the number of folds, how many folds are there in a gene or set of genes like the immunoglobulin heavy chains that might occupy 1% of the genome length in the germline but only 0.5% in the mature lymphocytes?  Comment on how this might have an impact on the frequency of development of autoimmune disease if a toxicant interferes with normal DNA repair enzymes.

The point is that DNA is so long (about 1 m, or 2 m if you consider that approximately half, the heterochromatic portion, of total DNA has not yet been sequenced, only the euchromatic portion has) it has to be folded many, many times to fit inside the nucleus (the computation should use the length of the DNA versus the maximum diameter of the nucleus) and that even a small stretch of DNA still contains many, many folds.  That folding means the DNA has to be opened up to allow transcription or replication. Somatic rearrangement eliminates a sizeable chunk of DNA and requires double-stranded repair of that area, a type of repair particularly prone to mistakes.  Not only does activation of a B or T cell lead to somatic rearrangement, it leads to active clonal replication, that is, lots of mitotic division.  And mitosis means more DNA cutting and pasting along with a surveillance on DNA repair that can be imperfect.  If imperfections of DNA arise as a result that are not eliminated by apoptotic mechanisms in the thymus, clones of these cells can persist. If these clones express anti-self immunoglobulins the result is an autoimmune disease.

Gut Physiology

20. The gut physiology of various mammals differs in ways that make oral intoxication of one species potentially very different from oral intoxication in another species.  Look up the comparative gut physiology for as many mammalian species as possible. Note differences in the sizes of the stomach, the length and nature of the small intestine, the presence or absence of a caecum and its size, the length and size of the large intestine and colon.  How might pure glucose ingestion affect a ruminant?  A hindgut fermenter like a horse?  Are there key differences among species that will define which toxicants will be more or less potent or efficacious in going from one species to another?  Are the test species (rat, dog, mouse, rhesus) most commonly being used good models for humans?  Or for other, wild species?

The point here is that gut physiology differs markedly across species.  As a result, considerations of pH sensitivity, impacts of gut microbes, and enterohepatic metabolism vary greatly across taxa and species.  The relative sizes and anatomical relationships of the segments of the digestive tract have significant impacts on toxicant absorption and elimination.  A toxin in one species may well not be a toxin in another species because of these variations.  And dose-response relationships may differ widely among animals.  Too much glucose in a cow drives the forestomach toward acidification and generates so much carbon dioxide the animal may suffer gastric rupture due to bloat.  In a horse the glucose also causes problems by altering hindgut fermentation.  Too rich a diet in a horse will actually cause starvation from calorie malnutrition (normally derived from partial digestion of cellulose in the hingdut).  Toxicants may have equally diverse impacts.  So a test species that differs greatly from the species of interest with respect to gut physiology may well be inadequate to provide good predictive data for the species of interest.

Macromolecular Chemistry

21. What are the chemical structures of cellulose, hemicellulose, pectin, glycogen, lactose, and sucrose?  Does this list contain the principle forms of dietary insoluble fiber or are there others besides the silicates and minerals, that contribute?

The structures appear in various sources.  The importance of insoluble and soluble fiber are also covered in those locations. Insoluble fiber acts to stimulate peristalsis and the movement of materials through the gut.  It also acts as an adsorptive surface to carry materials through the digestive tract and counters absorption from digesta by the gut.  Soluble fiber tends to slow things down and allow water and mineral resorption which is accompanied by increased toxicant and metabolite resorption.  Cellulose, lignin, and possibly hemicellulose fit into the insoluble category along with silicon dioxide (sand) and some minerals.  Pectin, gums, glycogen, and hemicellulose all fit in the soluble group along with additional minerals.  Lactose and sucrose are disaccharides (two simple sugars linked together) and do not contribute to either of these classes of fiber.

Do note here, again, that what begins as insoluble fiber in some species like ruminants does not end that way.  So what can be classes as fiber is physiologically handled differently across species and therefore may have differing impacts on toxicant exposures depending on what species is in question.

Portal Site Active Agent

23. In the compartmental models of pharmacokinetics much emphasis is placed on distributional volume and the role of toxicant sinks and sources within tissues.  Route of exposure plays an important role as does mode of elimination.  What happens to these models and considerations if the agent is active within the tissues where it is introduced?  For example, how do we deal with a compound that acts on the lung if it is introduced as an aerosol?

I refer you to the coverage in C&D on hepatic and lung, especially in chapters 5 (pp 116-117), 7, 13, and 15 (15 is not required reading).  My point with this question is that if a toxicant acts in a deleterious manner on a tissue that is important to its absorption or elimination it will very clearly alter the observed pharmacokinetics.  If the compound killed the liver cells that were involved in detoxifying it, the circulating half-life of the compound would be prolonged.  If a toxicant required an active transport process to be absorbed but acted to kill the cells involved, it would limit the absorption of the compound.  Note that in both these instances doses that were sublethal would display pharmacokinetics that are different than what happens at higher, directly toxic, doses.

Consideration of an aerosol delivery in the lung requires knowledge of the size of the aerosol particles because larger aerosols are eliminated higher in the alveolar tree.  Large aerosols act principally in the gut because they are moved to the digestive system in the nasopharyngeal area.  Those of intermediate size (2-5 um) are transported in a retrograde manner back to the nasopharynx from the bronchiolar tree.  Those less than 1 um enter the alveolar gas exchange areas where absorption into the blood occurs directly.

Damage high in the respiratory tree may well allow access of the toxicant to the bloodstream but would do so more slowly (as it requires the toxic insult first) than an aerosol acting within the alveoli.  Indeed, a toxicant acting to scar the alveoli might limit the uptake of the compound.  More acutely, however, it might simply increase the speed of toxicant absorption unless it compromised the alveolar capillaries during its actions.

Carcinogens and Risk Assessment

25. The statement is made in Klasssen & Watkins III, Companion Handbook for Casarett & Doull's Toxicology, 5th Ed. (p 189) that: "For risk analysis, it is assumed that cancer induction differs from all other toxicological events in that the induction of cancer is a nonthreshold phenomenon or an accumulation of many such irreversible events."  Is this justified mechanistically?

Obviously, based on an earlier response from an EPA employee, the EPA wonders about the same question.  Repair mechanisms may well mean the statement made is technically false.  From a practical, regulatory point of view, which is, by its nature, conservative, the statement is an attempt to optimize risk assessments.  Risk analysis works from data, not just biologically based models.  In dealing with cancer, it is dealing with information that is often qualitative.  Yes, there is a cancer; no, there is not a cancer with "yes" and "no" defined by agreed upon appearance of cells on histological slides or growth of abnormal masses on tissues.  Biological tissue will demonstrate a finite background of such abnormalities due to its innate growth properties or to exposure to agents that are not being controlled in a particular toxicological study.  So, from a theoretical viewpoint, there will normally be variation in the data for zero dose as well as every other dose tested.  Given that situation, there should again be a region of low dose where the biological response is not statistically different from that seen with the zero dose.  Analytically, we may or may not be able to measure residues that differ from zero within these animals, especially when we consider that there may be a very long latency between exposure and development of tumors.  So in this situation, we may be forced to look only at dose administered versus tumor formation.  The time lapse and numbers of animals that may need to be examined to distinguish background from a LOAEL, however, may preclude accurate establishment of an NOAEL.  Here again we would be forced to use a no threshold model.

Risk assessment also is usually looking beyond the induction stage of cancer formation.  Tumors are "fixed" and forming clonal expansions of the original transformed cell by the time they are detected by gross inspection and are usually at that stage when demonstrated histologically.  Though they need not be to the metastatic stage, they are usually beyond the point that repair processes can cause them to spontaneously involute.

Given these conditions, it is probably acceptable for risk analyses to make the simplifying assumptions stated.

Genetic Toxicology & Evolution

26. If pyrimidine dimers and chemical adducts to DNA are preferentially removed in transcribed and active DNA sequences relative to untranscribed and inactive DNA sequences, what does that suggest about the hot spots for genetic drift upon which evolution is based?  If you were looking for gene sequences to use to differentiate among related species, what kind of genes would you tend to look at as indicators?

If repair concentrates on actively transcribed regions (presumably because these are the most important for maintenance of life), then the untranscribed regions should be the ones that accumulate the most mutations. Indeed the whole idea that the Y chromosome is shrinking over evolutionary time is based on the evidence that it contains relatively few genes, most of which do not seem to be central to most of metabolism or cellular physiology.  While existing DNA sequences for many genes in many species allow comparisons using a large number of proteins, we might expect the most marked differences to exist between proteins that are relatively rarely transcribed.  So hemoglobins or cytochromes are probably not the genes of choice.  Rather protein markers of extreme age or crystalline proteins of the lens of the eye might serve as better choices.

Genetic Toxicity: Possible Models?

27. Why might the Plains Viscacha of South America be a good model for toxicity associated with oogenesis?  Could the DAZ1 gene found on the long arm of the Y chromosome be used as a screen for germ cell toxicity in the primate male?  How might the latter be evaluated?

Most people located our chinchilla relative and figured out that the female produces a remarkable number of eggs at each ovulation.  Indeed, it is not clear that the viscachia shares the same meiotic arrest or attretic processes that occur in most other mammals.  That she can only successfully carry twins is quite interesting in itself.  But she might well be a great model for examining preovulatory toxicant insults.  Post-fertilization might also work well at least until the time of implantation after which the viscachia's own physiology would probably complicate the toxicant evaluation process.  Note that there may be practical reasons why this species has not cropped up in the toxicological literature: the gestation is about as long as a small ruminant or monkey, the animal is not very small 2-8 kg, and is social, living in groups.  Housing groups of these animals would be expensive.

DAZ1 is so frequently deleted in human male offspring that it is unclear if these de novo deletions occur only in genetically susceptible families or if they exist in every male's spermatogenic products.  Since sperm can be evaluated for genetic mutations by lysis of the sperm heads and in situ hybridization using fluorescent DNA probes, it should be possible to screen semen specimens for DAZ1 deletions in combination with Y chromosome specific DNA markers.  If germ cell toxicity would be evident as an increase above baseline in those Y chromosome bearing sperm that lack the DAZ1 marker.

DAZ1 is conserved across primates making it a good tag for the insults suggested.  However, it is also present as a six-fold tandem repeat which means that up to five copies could be deleted without losing a positive in situ hybridization signal for the locus.  Thus, to use the marker optimally, some kind of intensity index would be needed that could demonstrate how many copies of DAZ1 existed in a given sperm.

Alternatively, quantitative PCR methods could also answer the same questions.

Cross System Enzyme Induction

29. A number of endogenous compounds as well as xenobiotics cause induction of enzymes that apparently have no role in their own metabolism.  Why might this make sense evolutionarily?  Or, why might it simply be a relic of evolution within these metabolic systems?  Provide specific examples, if possible, to defend your argument (or demolish someone else's)

Induction of phase I and phase II enzymes are the classic examples here.  The plethora of CYP genes, however, probably arise by repeated gene duplication, translocation, and divergence.  It may well be that control elements that were important for metabolizing one class of compound remained with a divergent enzymatic gene making the control an evolutionary relic.  On the other hand, coordinate control of diverse metabolic pathways, e.g., electron transport systems to produce ATP and sodium pumps in the cell membrane that use ATP, can often be found.  Since many of the CYP enzymes use NADP(H) as a cofactor for activity, coordinate regulation of their activities makes metabolic sense.  The involvement of amino acid metabolism and glutathione production and use also make coordination between glutathione use and production a probable arena of coordination.

Assignments for Session 3: Calcium Controls

2. Describe the control circuit for parathyroid hormone, calcitonin, and cholecalciferol; how would a blocker of 1[alpha]-hydroxylase impact calcium deposition in bone?

This is simply a review of calcium and bone metabolism.  I refer you to any endocrinology, physiology, or cell biology textbook. The pertinent slides exist near the end of the list on my Endocrinology course website.  Parathyroid hormone (PTH) and calcitriol (1,25-dihydroxy-cholecalciferol; 1,25-dihydroxy-vitamin D3) counter the actions of calcitonin (CT).  PTH and calcitriol cause serum calcium levels to rise by increasing calcium uptake from intestine and bone; CT lowers serum calcium levels by stimulating calcium clearance through the kidney and blocking reuptake from bone.  Further, calcitriol is formed by the sequential actions of UV light on 7-dehydrocholesterol passing through exposed skin, liver 25-hydroxylase on cholecalciferol, and 1-hydroxylase in kidney on 25-hydroxy-cholecalciferol.  The 1-hydroxylase is stimulated by PTH while a competing, inactivation pathway involving 24-hydroxylase is stimulated by CT.

Note that in the human most of the control is exerted via the PTH and calcitriol arm of the controls with CT playing a normally minor role.

A 1-hydroxylase blocker would tend to block bone resorption and therefore promote calcium deposition in bone.

Discussion Questions

PCBs & Thyroid Function (QS2Q3)

13. Exposures to PCBs can potentially alter thyroid function in part because of the structural similarities of some of the PCB isomers and thyroxine or triiodothyronine.  About 50% of thyroxine is carried in circulation by thyroid binding globulin, TBG, while 45+% is carried by albumin or transthyretin.  Much of thyroxine enters cells by diffusion, but a substantial quantity also enters via the aromatic amino acid transport proteins in cell membranes.  After cell entry, thyroxine is deiiodinated to triiodothyronine which then binds to receptors that usually reside on DNA binding sites within the nucleus; binding often results in release of the hormone receptor complex from DNA and in a change in DNA structure and transcriptional activity.  What are the obvious possible targets for PCB toxic effects?  If we include molecular clearance pathways, are there other targets?  What if the toxicant is a slightly acidic derivative of a PCB?  What about an amine derivative of a PCB?

<>Appropriate distinctions between carcinogenesis and genetic toxicity? (QS4Q5)

   Although cancer can arise in germline cells, it happens with higher frequency in other cell lines, i.e., it tends to be a problem arising primarily in somatic tissues.  On the other hand, the vast majority of post-fertilization losses during gestation and the inherited diseases linked to DNA mutations or chromosomal anomalies are the results either of inheritance via germ cell lines or due to genetic damage occurring during meiosis.  It seems that lumping these two very different types of conditions together under the rubric of genetic toxicology or genetic toxicity confuses a number of issues with respect to the mechanisms by which the conditions arise and the impacts these changes have on individuals and populations.  I suggest that it is probably better to speak of somatic cell genetic toxicity and germ cell genetic toxicity than to hold with the current dichotomy of carcinogenesis and genetic toxicity.  Do you agree or disagree?  Why or why not?  What evidence can you marshall to support your position?

© 2005 Kenneth L. Campbell