Session 1, February 3-7, 2003

Mammalian toxicology, Biology 666.

Assumptions in dose response, Possible case studies, Efficacy vs Potency Hide the lecture Assumptions in dose response, Possible case studies, Efficacy vs Potency. Edit information about the file Assumptions in dose response, Possible case studies, Efficacy vs Potency Permanently delete the lecture Assumptions in dose response, Possible case studies, Efficacy vs Potency and all of its contents. Notes: Session 4, February 14, 2002

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.

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 reisolated 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 (page 18 of the Handbook)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]

There is a molecular or receptor site (or sites) with which the chemical interacts to produce the response.

The production of a response and the degree of response are related to the concentration of the agent at the receptor site.

The concentration at the site is, in turn, related to the dose administered.

3. 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 postbinding 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 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.

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.

Possible Case Studies

1. DDT and persistent pesticide bans

2. Dredging of the upper Hudson for PCBs

3. Arsenic wells in northeast and eastern India and Bangladesh

4. Zero tolerance for Alar in apples

5. Labeling and restriction of milk from cows injected with or transgenic for bovine GH

6. Oral contraceptive contamination of municipal sewage effluents in Great Britain

These should take into account the following items:

A. Mammalian associations

B. How does toxicology play a role in these cases?

C. What are the assumptions, stated or unstated, that support the contrasting arguements in these debates?

D. Are risk assessments being used? If so, are these realistic and well grounded?

E. What particular elements of toxicity testing and modeling are being employed? Give examples.

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1. Dermal exposure

Dermal exposure shares many traits with SC but pores are also important routes for diffusion into the body. Structure of the skin includes not only the multiple dermal layers but the organization of the hair follicles which allow chemicals penetrating the follicles a much shorter diffusion route into the body than through the cornified exterior layers of the epidermis.

Dermal, mucosal, and inhalation routes are particularly illustrative of the impact of the delivery vehicle of a toxin or toxicant. Aggressive agents such as strong acids, bases, or phenol erode integumental layers and allow more ready access to vascularized interior layers; they shorten the diffusion path needed by an extraneous compound to enter circulation. Similarly nonpolar solvents may dehydrate external cell layers and allow more ready access of solutes to vascular space. Detergents which allow formation of micelles when present above their own solubility limit (critical micelle concentration) can promote movement of compounds through tissues by enhancing compound delivery to the lipid layers of the cell membranes or by carrying them as micelles via aqueous channels between cells. Detergents tend to soften and remove epidermal layers and open pores thereby enhancing toxin or toxicant movement across outer tissue layers.

2. 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 or procedures intended to look at the cost/benefit ratio of exposure versus nonexposure, 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 based on 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

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1. Mammalian Toxicology

Session 2, Feb. 7, 2002

Note the time change for class on T 2/12/02 & 2/19/02 to 1:00-2:30 PM. Note also the invitation to join me (KLC) at the Instructional Technology Center presentation on the Prometheus on-line course framework on T 2/19/02 from 10:00-12:00.

Beginning to address some questions posed in Session 1.

1) Why might a lethality (dose/response) curve plateau at a level less than 100%?

This is probably best explained by the existence of genetically determined individual variation of genes, gene expression, and protein production (expressed phenotype). Genetic variation may give rise to organisms that are not susceptible to a given toxicant either because they fail to express a protein target of the toxicant or because they possess degradative mechanisms that inactivate the toxicant. Paths to inactivation may be entirely new or they may simply be more efficient than those found in other individuals. Note that these same considerations will apply to cross-species susceptibilities, so some species will be sensitive to some toxicants or toxins while others will be much less susceptible or not susceptible at all. Examples here would be drug resistant strains of bacteria, or malarial parasites, pesticide resistant strains of mosquitos, or flies, or herbicide resistant strains of grasses.

2) What forms of dosage and exposure are there?

Total dosage may be less important than concentration but this depends on how acutely or chronically the dose is delivered, just how toxic is the toxicant, whether the toxicant is cleared or accumulated, and, whether, when delivered, the toxicant is in a form that is bioavailable.

The old saying that "The solution to pollution is dilution" has some truth to it in that many toxicant problems arise when man concentrates naturally occurring toxicants and exposes other humans, or other biotic or nonbiotic elements of his immediate environment to supranatural concentrations of these materials. Excellent examples here are the naturally occurring radionuclides used in research, medicine, and industry or the plant concentrates like cube resins used as pesticides in agriculture and home lawn care. If exposure occurs at a low level over a long enough period of time for the toxicants to be deactivated either by metabolism or adsorption to surfaces (making them unavailable), the risk of a toxic response may be low. If the same total dose (amount) is given over a short term, however, toxic response will be likely as the exposure overwhems inactivation or adsorption capacities.

Form of delivery of the toxicant is also an important factor in determining biological response. For lipophilic materials, adsorption on hydrophobic surfaces such as charcoals or clays will often make the toxicants unavailable to biological systems unless extremes of pH or mechanical agitation liberate the toxicant materials as they pass through a particular digestive system. Dilution of lipophilic materials in oils prior to introduction into a mammalian system will tend to slow release of the material to the biological system because of the physico-chemical interactions of the lipophilic solute and solvent. Charged molecules like cations may also adsorb strongly to certain zeolite clays or to humic acids in light soils. On the other hand, such materials should be readily released from aqueous suspensions.

Related issues associated with bioavailability arise when a compound is bioaccumulated following administration. Persistent hydrocarbon pesticides and PCBs are excellent examples of how lipophilic toxicants can accumulate during chronic exposure largely based on the physicochemical characteristics of the molecule in question. By partitioning into lipid droplets or biological membrane lipid layers these compounds become sequestered against biological breakdown and against biological response until they reach such levels that the fats within an animal cannot accumulate any more and/or the degradative systems become saturated so levels of the toxicant in aqueous compartments rise and evoke toxic responses. Note that fluctuations of animal adipose levels may release bioaccumulated toxicants during lean periods and reduce toxicant risk during periods of abundance. Declines in body fat levels often coincide with the breeding and gestation periods of many birds and mammals. Since fat is the most concentrated form of biological energy available, the embryo in all species is provided a yolk source for early development and in mammals is provided milk which also contains high proportions of fat. Because of fat mobilization during gestation and lactation many animals including birds, reptiles, and mammals naturally expose developing young to higher levels of bioaccumulating lipophilic toxicants than the adults are exposed to. As a great deal of the lipid delivered to the young is used in energy or macromolecule production, toxicants often become available in concentrations sufficient to elicit biological responses.

Likewise materials that can accumulate in bone, such as some ions, will also tend to be sequestered during chronic low level exposures. Since gestation and lactation mobilize the elements of bone, toxicants that are hydrophilic may also be transferred in biologically effective concentrations to developing embryos and to nursing offspring.

Evolutionarily, how might the downloading of toxicants to offspring be useful or unfavorable?

Routes of toxicant/toxin delivery include: sc, subcutaneous; im, intramuscular; iv, intravenous; ip, intraperitoneal; po, per os (via the mouth); inhalation; topical/percutaneous; and transmucosal.

Biological responses arising from a single dose at a single concentration delivered in these several ways may well differ because of the differences in rate of absorption of the dose from the site of deposition, the availability of sites for adsorption of the administered dose, the presence of degradative enzymes in circulation or target tissues, the presence of unusual or extreme conditions (e.g., stomach pH), and the rate of transit from the site of administration through the biological system and back to the environment (e.g., rate of respiration for a volatile toxicant, or rate of gut transit).

A review of blood circulation http://www.ultranet.com/~jkimball/BiologyPages/C/Circulation.html and lymphatic circulation http://www.merck.com/pubs/mmanual_home/illus/167i1.htm or http://www.bioanim.com/CellTissueHumanBody3/limfVse1lgws.html is suggested to understand how these various routes of exposure may differ so greatly.

IV is the most direct route, but response may differ if injection is done above the heart or below it as much of the lower circulation encounters the hepatic portal circulation which carries it through the very active hepatic degradative enzyme systems. Some compounds, e.g., estradiol are readily degraded in the liver and do not survive passage except by adsorption to carrier proteins like albumin. On the other hand, volatile compounds may survive poorly if injected in the upper extremities due to passage through the pulmonary circulation with losses occurring in the lungs.

IP passes via the lymphatics or the mesenteric venous drainage to the circulatory system. The lymph systems deposit injected materials into the venous drainage above the hepatic portal system while the mesenteric vein empties into the hepatic portal vessels.

SC involves uptake by the lymphatics or by diffusion into the venous drainage of the introduction site.

IM tends to be slow delivery as lymphatic drainage tends to be less important than diffusion through the muscle tissues to blood vessels.

Transdermal/topical/percutaneous exposure will rely on much the same delivery mechanisms as SC, however, the barrier of the skin must be breached either directly or via diffusion, often in the presence of a carrier solvent.

Transmucosal exposure will be similar to transdermal or sc, except that the vascularity of mucosal membranes will tend to favor vascular drainage of the exposure sites.

Inhalation exposure will involve diffusion into the pulmonary venous drainage of the lung. Although some degradation may occur in the lung, much clearance will occur either in the liver or the kidney.

PO exposure may involve transmucosal adsorption from the mouth or GI tract. But it may also involve chemical modification in the stomach acid. Depending on the chemical involved, this may degrade the compound, hydrolyze it and make it more easily degraded or more easily absorbed, or protonate or hydrolyze it so as to make it less readily adsorbed.

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What is toxicology?

Definition: Study of deleterious effects of chemicals

Comparison to Pharmacology (Study of medical properties of chemicals) or Endocrinology (Study of physiological responses to endogenous chemicals)

What are toxins?

Naturally occurring compounds that may demonstrate deleterious effects when introduced into another organism.

What are toxicants?

Compounds of anthropomorphic origin that demonstrate deleterious effects when introduced into an organism.

Some considerations of historical background:

The classical Greeks started classifying those poisons that they encountered in conduct of their daily lives as being derived from mineral, plant (& fungal), or animal sources.

Important examples of toxicants that the ancients might have encountered include heavy metals (Hg, Cu, Pb, Sn) or homologs of S such as Ar. These are all found abundantly in minerals and soils from volcanic areas and are elements released in the heating of ores during refinement of metals. During the Bronze age this would have been common. Arsenic is also found in soils such as those in India and Bangladesh where it can be leached into drinking water.

Examples of toxins would occur in plants, fungi, and animals. The poison ivy family (poison ivy, poison sumac, poison oak, cashew, shellac, mango...) all produce a mixture of reactive oils called urushiol. This can cross-link and otherwise modify self-proteins resulting in generation of an immune response. Presence of immune memory often results in an anaphylactic type response upon repeated exposure (so long as the offending urushiol is allowed time to be absorbed and to react with proteins in a manner similar to the initial sensitizing exposure). Hemlock, nightshade (including the precursors of tomato, potato, and sunflower), jimpsom weed, and others contain chemicals toxic to potential plant predators. Many large fruited plants including apples, peaches, and apricots have seeds encased in very hard coverings within otherwise very edible, fleshy fruit. The seeds often contain cyanoglycosides which generate HCN when digested in stomach acid. Again, the toxins appear to be deterrents to predation, this time of the seeds. Fungi related to the Amanita mushrooms as well as other varieties also produce toxins. For plants these toxins appear to serve as defense mechanisms. (A toxicology of plants is found in: http://www.fao.org/docrep/x2230e/x2230e15.htm )

Animals such as poisonous snakes, arachnids, many mollusks (including jellyfish, cone shells, and some ocotopi), and certain insects like wasps or hornets use toxins both for protection, and, more importantly as a means for food procurement. The array of tissues affected in mammals by the venoms of snakes, arachnids, and mollusks is very large. It includes the CNS, peripheral nerves, respiratory centers, voluntary muscles, vascular endothelium, specific blood cell types, heart, kidney, immune system, among others. Likewise the manner in which the toxins from arachnids or fish or mollusks act on insects or other fish is similarly broad.

These classifications make sense for the ancient world. Many of them would be known to shamans and other traditional healers or keepers of cultural history, tradition, and lore.

But what about bacterial toxins, as the ancient world would not have known of these?

The endotoxins produced within an organism following infection by a bacterium non localized to the digestive tract or external integument are responsible for many of the symptoms associated with these disease conditions. Again, they may affect a wide variety of systems and may do so by a wide spectrum of means which may include impacting immune function, continually activating signal transducers involved in endocrine or nervous signal transduction, or inhibiting the elongation factors involved in cellular protein synthesis. Bacteria such as the "O" strain of E. coli, Clostridium botulinum, Staphylococcus aureus, Bacillus anthracis, Vibrio cholerae, or Corynebacterium diphtheriae all act as pathogens by generating potent endotoxins within infected hosts.

What about viruses?

Viruses or certain bacteria may induce their hosts to produce toxins. Vibrio do not generate toxin unless they are infected by certain bacterial viruses. The Red Tide is generated by algae that have become infected by certain bacteria.

What about prions, the infective agents for slow viruses? Are these toxins in an of themselves as they directly induce a deleterious consequence in an infected host?

During the late middle ages and into the early Renaissance the idea of evaluating the impacts of potential poisons arose. Looking at what organs were effected by a poison became an important idea as did evaluating the impact of a poison on a test subject, e.g., a test on a dog might be done before applying a poison to a human. Paracelsus also stated the central tenant that effect was proportional dosage. Not only did this play a central role in formulating toxicology, but it formed a pillar of pharmacology and endocrinology as well. Moreover, the idea that a deleterious substance might be less deleterious if given in a small dose and, in fact, possibly even beneficial, ultimately gave rise to the train of thought leading to part of homeopathic medicine.

What is homeopathic medicine? Holistic medicine? Allopathic medicine?

How do these forms of medicine approach the topics of toxicants, toxins, and toxicology?

Paracelsus' idea that responses to chemicals, toxins, or toxicants was graded ultimately leads to the idea of a Dose-Response curve. This is an underlying tenant of endocrinology, pharmacology, and toxicology. The normal curvilinear response reflects the underlying enzymatic machinery responsible for "running" each cell. It is the cumulative sum of the activities of the enzymes within any given cell and the summation of the activities or responses of each single cell within a tissue, organ, or organism. For single celled organisms, or for measures of whole organism response such as lethality, it is the population response. Again, a cumulative sum of the activities of each individual cell or organism and its component parts.

For a given response, say a lethal response, the curve rises from a basal nadir asymptote along a smooth curve to a maximal asymptote. The maximum results from saturation either of cellular systems or of a population, e.g., 100% deaths. The inflection point of the curve falls at 50% of maximal response. The dose corresponding to this level of response is termed the LD50 in lethality assays or the ED50 in assays of nonlethal response.

Would it be possible to have a lethality assay in which the maximal level of response was less than 100%? Why?

In assessments for nonlethal response, the dose response may be biphasic, rising from a basal low value (rising response) to a maximum peak or plateau, and then declining at higher doses back toward or beyond baseline. Thus, the low dose end of the biological response to the chemical may be positive, e.g., palliative, as in pharmacologic or endocrine compounds, while the high dose end is negative or toxic. The distance between these regions of response constitutes the margin of safety for a particular drug or a chemical used for purposes other than as a poison.

Toxicology includes consideration of the factors that determine margin of safety, or relative risks associated with exposure to a particular toxicant or toxin.

When we say "dose," what kind of dose is referred to? What are the routes of exposure to toxicants and toxins? What physiological barriers stand between a toxicant and its ultimate cellular target(s)? Does the form of the toxicant play any role in exposures or penetration of physiological barriers?

Is it possible to have a threshold value for a toxicant below which there is no effect? What are the reasons or mechanisms that might underlie such a threshold if it does exist?

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Project ideas, case study possibilities. What about Hanford radiation ground water contamination case? Suggestion was, in such situations, to 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 focussing 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 Airforce 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, meatpacking, 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 .

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 Cassaret 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.

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.

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Session 10.

Projects:

Allison: Now organizing information, about 10 citations so far with expectations of 3-4 times that many.

Neetu: Savannah River, tritium & plutonium contaminations; Techa River, cesium contamination.

Joanne: Target medications likely will be ACE-inhibitors, Dilantin, and lithium; looking at effects during gestation both on the mother and the conceptus.

1. Review of comparative gut physiology across mammalian species.

The presence of major fermentation compartments in foregut and hindgut fermenters makes these actually symbiotic ecosystems rather than individual organisms. Elimination of the fermentative organisms in foregut fermenters would starve the host animal. In hindgut fermenters it would force them to increase forage intact, potentially to the level of malnutrition. In many other species it would cause diarrhea but not necessarily lethality.

The absence of a gall bladder in rats causes differences in the manner and rates of bile production and the exposure of the bile contents to enterohepatic recycling.

The length and infolding of the small intestine determines the effective absorptive surface area and the potential for absorption of toxicants in the gut compartment. Similar considerations also occur for the colon.

2. The question arose as to whether, given the cautions regarding food additives or composition across species as complicating issues in toxicity evaluations, whether, as claimed in some lay literature, microwaved food was a hazard during pregnancy and gestation.

Evidence is slim that the limited microwave exposure caused by waiting outside a microwave would have a direct effect on mother or fetus. Moreover, save for heat, the microwave, unless prolonged will not leave a residue in the food that might make such food hazardous. Rather, it is more likely that the suggested avoidance of microwave foods during pregnancy is more based on their typically higher content of salt and preservatives as nutritionally less adequate than any exposure of the food or the cook to microwaves.

But this raises the more general question of whether irradiated food, e.g., gamma irradiated meat or milk, is safe for consumption?

It should be safe because the irradiation is in the past relative to presentation to the consumer. Radiation does not leave residues or residual radiation in the food. Moreover, the act of irradiation kills the degradative organisms in the food that could cause formation of noxious or toxic products; it should make the food safer rather than less safe. Finally, empirical evidence, lack of negative or untoward effects in areas where irradiated food is used suggests such food is safe.

Is there anything that might make it unsafe? Only if the irradiation is sufficiently strong or prolonged to cause chemical changes that generate toxic byproducts in the food. More likely such food would become physically "burned" or otherwise undesirable in addition to becoming "less safe." Note there is little if any empirical evidence indicating that such byproducts are encountered during the routine processing of foods for consumer consumption.

Such questions raise the points of health concerns during radioisotope use, i.e., radiation health physics (RHP) concerns. RHP is concerned with minimizing the exposure of subjects to the electromagnetic and particulate emissions from radiation sources. Note that both of these types of radiation primarily impact targets or subjects by delivering energy: in the case of x-rays (arising from electron transitions in decaying radioisotopes, often a fall from a high energy outer orbital into a low energy inner orbital accompanied by the release of a corresponding photon of high energy electromagnetic radiation) or gamma rays (arising from transitions within the unstable nucleus, e.g., conversion of a neutron to a proton and a beta particle (energetic electron) often with the loss of a photon of high energy electromagnetic radiation) the energy delivered is in the form of high energy photons that can cause chemical bond alterations including breakage and/or molecular motions disruptive of normal molecular function (e.g., in biological systems). Particulate emissions like beta particles, positrons (positively charged electrons), alpha particles (2 neutrons + 2 protons), or neutrons can impart kinetic energy to target molecules causing either movement or bond disruptions. If particle bombardment is intense enough, the larger particles like alphas or neutrons, especially, can also cause nuclear alterations that can generate secondary radioactive atoms, e.g., chlorine. Thus, in either case energy is being imparted to the target and may result in alterations, some deleterious, to the target. Particles, however, in colliding or glancing off the electron shells of the atoms of the target, rapidly lose energy and, with the exception of the neutron, do not penetrate efficiently within a target medium. Neutrons can penetrate because they lack a net charge, they have no intrinsic tendency to approach or be repelled by the electrons, or protons, of target molecules, they are unaffected by the atomic magnetic fields and do not affect those particles or fields unless they undergo a direct collision with a nucleus or electron. (Photons penetrate better than charged particles but have an intrinsic electronic and magnetic character that causes them to interact with electrons and atomic magnetic fields; when the allowed energy transition levels of target molecules match those of the photon moving through the target medium, those photons are efficiently absorbed.)

Recapping the electromagnetic spectrum: long wavelengths mean fewer photons per unit time through a linear distance, lower frequency, lower energy (wavelength is inversely proportional to frequency. Radiowaves with wavelengths of >um (up to m or km) occupy the long end of the spectrum, followed by microwaves (um lengths), infrared (IR, heat, ~800 nm - a few um), visible (300-800 nm), ultraviolet (UV, 10's-300 nm), x- and gamma-rays (angstrom - nm), cosmic rays, and intranuclear forces (atomic/subatomic dimensions).

Note that since radiation is propagated from the source in all directions the flux of that radiation across any plane placed near the source will be inversely proportional to the square of the distance from the source (distribution in two dimensions, not just one). Thus, several of the rules of RHP have to do with maximizing the distance or effective distance from the source.

The three cardinal rules of RHP are:

1. Minimize time of exposure

2. Maximize distance from source

3. Optimize shielding (protective clothes, gloves, shields, behaviors)

Note that 3. is actually a variant of 2. in that it addresses "effective" distance from the source. Thus, if a radiation absorbing shield is interposed between the source and the subject, we are essentially increasing the effective distance between the source and the subject. Note also the indication of optimizing shielding. Although it might be intuitive to use a high density shield which normally corresponds to a shield made of a material of high atomic number, we should be aware that such materials provide larger atomic targets for some forms of radiation, particularly highly energetic particles. When such highly energetic particles, e.g., the beta particle from 32P decay, encounter large atoms (e.g., in lead or leaded glass shields) they often cause electron orbital alterations that result in the production of secondary particle (Compton electrons) or x-ray emission (Bremstrallung). As the electromagnetic radiation is more penetrating than most particles, the secondary emissions may prove more damaging than the primary radiation. Thus, in such cases, low to moderate sized atomic nuclei are used to build composites that can then be used as effective shielding, e.g., high density plastic (carbon/oxygen/hydrogen) or aluminum.

Now note that these same rules for limiting radiation exposure have close or exact analogs in rules to limit chemical or toxicant exposures:

1. Minimize time of exposure. (This limits the dose and/or the time for the toxicant to bind to its target molecules or to act on its target molecules.)

2. Maximize distance from source. (This minimizes the dose or encountered concentration.)

3. Optimize shielding. (This limits the routes of exposure and/or optimizes the actions of endogenous barriers, e.g., the immune system -- vaccines, or repair processes.)

Close the folder Session 11. Pharmacokinetics. Folder Session 11. Pharmacokinetics Hide the lecture Session 11. Pharmacokinetics. Edit information about the file Session 11. Pharmacokinetics Permanently delete the lecture Session 11. Pharmacokinetics and all of its contents. Notes:

Session 11.

Projects moving slowly ahead.

A review of math and scaling units (http://www.onlineconversion.com/)

Comments on comparative digestion: note that humans resemble omnivores like pig and rat more than they do ruminants like sheep or hindgut fermenters like rabbits. Accordingly, the metabolism of toxicants will differ considerably from such animals. Indeed, the diversity that allows optimal occupation of environmental food niches is a real stumbling block to modeling the toxicokinetics or pharmacokinetics of exogenous chemicals. There are variations in digestive tracts that are emblematic of the variations in other systems. These mean the movements of chemicals through these systems, the actions of the systems on the chemicals, the actions of the chemicals on the systems, and the removal of the chemicals from these system will differ among taxa even if not remarkably among species. Handling of carbohydrates in foregut and hindgut fermenters as opposed to those with primarily forestomach digestion is highly dependent on intestinal fauna and pH. Forage quality can have marked impacts on these parameters (e.g., Ghosts of the Past, ). Ruminating or foregut digesting as opposed to hindgut digesting will also have important impacts on the size and types of particulates present in the absorptive areas of the intestinatl tract. As noted previously such particulates, including cellulose, lignin, pectin, and silicates, can act to limit absorption of toxicants if they are relatively clean, or they can release such toxicants if they are previously saturated with them. (Note Neetu's response to a question on carbohydrate polymer chemistries as it contains good links demonstrating these structures and the small changes in structure, alpha versus beta linkages between glucose residues, that make some molecules like glycogen digestible with normal mammalian enzymes while others like cellulose require the enzymes found in certain bacteria or fungi.)

Pharmacokinetics

Define:

What is considered under this topic?

Not only route and concentration of dose, but also volume of distribution , bioavailability, perfusion and diffusion. The manner in which these play a role is often determined by which model of physiological distribution is most appropriate to the compound in question.

State assumptions for 1 compartment and 2 compartment systems.

1 compartment system: body is a homogeneous unit

2 compartment system: body is an inhomogeneous unit

In the 1 compartment system the clearance kinetics resemble a simple half-life plot as in radioactivity, where constant proportions of chemical are lost over unit increments of time. The plot of log tissue concentration versus time is a straight line with a single negative slope.

In the 2 compartment system the clearance kinetics have two or more phases and a plot of log tissue concentration versus time after dosage can have a rising, or loading, limb and a falling, or unloading, limb.

Note the comparments are graphical or mathematical constructs that may or may not have a straight forward correlate within the biological system. An example of the different loading and unloading rates observed when 125Ialpha/131Ibeta hCG is administered to the female rat. Plasma levels tend to be parallel for both isotopes with a rising and falling limb, but there is a non-parallelism that is evident for the two isotopes as they past into the kidney. This demonstrates the physical existence of at least two compartments for the processing of the two subunits of this glycoprotein molecule in this physiological system.

Apparent distribution volume:

Actual distribution x affinity in distribution sites and/or metabolism in those sites may yield a calculated distribution volume that is greater than vascular (or even body) volume. In the simpliest case such as inulin which is not metabolized and does not penetrate beyond the vascular space:

(# of counts of administered radiolabeled inulin/"x = total vascular volume") = (measured counts/measured plasma volume)

and x then gives the actual total vascular volume. For other compounds the distribution volume will the > or < the total vascular volume because the compound may be bound or sequestered and held out of circulation or it may be cleared and thus removed from circulation.

Close the folder Session 12. Orders of Magnitude: What environmental toxicant levels are relevant?. Folder Session 12. Orders of Magnitude: What environmental toxicant levels are relevant? Hide the lecture Session 12. Orders of Magnitude: What environmental toxicant levels are relevant?. Edit information about the file Session 12. Orders of Magnitude: What environmental toxicant levels are relevant? Permanently delete the lecture Session 12. Orders of Magnitude: What environmental toxicant levels are relevant? and all of its contents. Notes: Session 12.

Prometheus problems: care and feeding of URLs, posting of images

UMB Mass Media Article: Cohen, M & N. Natelson, "Animals Don't Belong in Chemical-Testing Formula." The Mass Media, March 7, 2002, p.22. Please read and formulate a responsive comment. You might find the following link helpful http://www.chai-online.org/chai_is.htm

The following AP Wire Article also appeared (text is in the file attached above): "Waterways awash in medicines, personal care products," The Providence Journal, March 13, 2002, page A5.

This is a summary of an article from a study by the US Geological Survey published in the March 15 issue of Environmental Science and Technology. It mentions chemicals from over the counter medications and personal care products as being present in drinking water samples as in the "parts per billion" range. What does ppb actually mean chemically and physiologically?

Similarly contaminants are sometimes listed as in the ppt (parts per trillion) or more commonly in the ppm (parts per million) range. Where do these fit along the scale of concentrations normally encountered in physiological chemistry?

The suggestion that charcoal might be used as one form of effective additional water purification was discussed because of its effectiveness in removing hydrophobic materials, including most

organics from solution. Another alternative involving dialysis membranes is discussed in the following report: http://www.cenorm.be/standardization/tech_bodies/workshop/otherthanICT/ws1/resources/cwa0 01_ad.pdf .

Also note the following site as a good prospect for reviewing basic material covered to date:

http://www.biology.arizona.edu/chh/problem_sets/toxicology/toxicology.html

File APartonperscarepollut.doc (27.5 KB) Hide the file APartonperscarepollut.doc. Edit information about the file APartonperscarepollut.doc Delete the file APartonperscarepollut.doc. Close the folder Session 14: Carcinogenesis: Problems with Some Aspects of Coverage by Casarett & Doull. Folder Session 14: Carcinogenesis: Problems with Some Aspects of Coverage by Casarett & Doull Hide the lecture Session 14: Carcinogenesis: Problems with Some Aspects of Coverage by Casarett & Doull. Edit information about the file Session 14: Carcinogenesis: Problems with Some Aspects of Coverage by Casarett & Doull Permanently delete the lecture Session 14: Carcinogenesis: Problems with Some Aspects of Coverage by Casarett & Doull and all of its contents. Notes: Session 14:

Prometheus issues:

-- Access problems, off campus requires a PIP link (unfortunately true for all communications to the university sites)

-- Project postings by students, where do we put them? [This is a question that has been posted to the support site for Prometheus. We are awaiting an answer. At present it may be best to post responses to my note in Discussion or to send them in as Messages that can subsequently be reposted if needed by the instructor.]

Carcinogenesis:

Why screen breast cancers for estrogen sensitivity if hormones are not directly involved in initiation of neoplastic growth as suggested by Casarett and Doull?

Breast tumors are screened for the presence of estrogen receptors because estrogen acts through such receptors to promote expression of those genes that lead to cellular replication via mitosis. Estrogen is promitotic in many or the tissues in which it normally acts. Thus, knowing if a tumor is sensitive to estrogen can provide a means to suppress tumor growth. Aromatase inhibitors can be given to suppress ovarian (if a premenopausal woman is involved) or peripheral production of estrogens. This will often slow the tumor growth or its production and response to paracrine growth factors like EGF, or TGFbeta. In turn, this provides more opportunity to apply radiation, surgery, and/or directed chemotherapy agents to ablate the tumor. Obviously, if the tumor does not have estrogen receptors, it will not respond to such treatments.

Likewise, testosterone or dihydrotestosterone are promitotic in male secondary structures like the prostate. Here antiandrogens can be applied to good effect so screening could be used for some tumors of the prostate. (Normally slow progression of these growths and the lack of ready anatomic access to the prostate tends to make this a less frequently used approach.)

Casarett and Doull point to the actions of growth hormone and other growth factors and suggest these may be considered chemical carcinogens. This is contrary to the definition of toxicants and really only fits the notion of toxins except that both refer to substances administered exogenously. Certainly hormones are not exogenous. Their actions at the high levels characteristic of endocrinopathies may eventuate in neoplastic conditions. Whether they are truely the initiators of these neoplasias can probably be questioned (environmental factors, exogenous radiation, etc.). Model systems such as knockout or overexpression models do, however, suggest that hormones can, if not initiating cancers, at least act as sensitizing or predisposing agents. Such is probably the case for the elevation of dimethylbenz[a]anthracene (DMBA) tumor formation seen in rats expressing high levels of prolactin. Both estrogens and prolactin are mitogens in rat breast tissue. Their actions will increase the number of cells undergoing division in any given time. If DMBA is applied simultaneously, or subsequent to this action, the intercalating agent DMBA will have additional opportunities to disrupt the crucial machinery involved in DNA synthesis and repair associated with mitosis. That does not make estrogen or prolactin cocarcinogens, but it does mean that they will predispose responsive tissues to agents that interact with DNA. The same situation will also apply to dimethylnitrosourea application to prostate tissue simultaneous with or subsequent to promitotic androgens such as testosterone. In fact, it should apply in any situation where a hormone alone stimulates a tissue to increase mitotic activity.

The text authors also seem to hold a somewhat dated understanding of how hormones and genes play roles in neoplastic and cancer development and growth. I would definitely refer students to the publications of Drs. Robert Weinberg of the Whitehead Institute at MIT and Polly Matzinger of NIH with respect to critical factors involved in the processes of cellular transformation and immune surveillance associated with cellular transformation, respectively. Several pertinent files or articles are linked to these lecture notes.

Weinberg in a lecture to the Endocrine Society last summer in Denver, mentioned at least four cellular features that had to be altered before a cell could undergo transformation from a normal to a cancerous cell. First, mutations or alterations had to occur in one or both the the cellular mitotic checkpoint proteins, pRb, and p53. Second, a change in expression of proteins involved in the apoptotic pathways had to occur so that this process was either not initiated or was actively blocked. Third, telomerase had to be expressed so that the cell could overcome the limitation on division normally encountered due to shortening of the telomeres in each round of cellular division. Normal differentiated cells express little or no telomerase. Transformed cells express significant levels. Finally, to actually form a tumor, the transformed cell has to avoid immune surveillance.

The latter point is probably best addressed by Matzinger's work which posits that the immune system is not active as normally thought, but rather, reacts only to the products of cell death. Either released cytokines or chemicals that ultimately cause production of immunoattractants like leukotrienes or interleukins.

Note that the complex of steps required for expression of full-blown carcinogenesis means that the frequency of truncated events that fail to reach this level is probably considerable even under normal circumstances. The elevations in this basal rate is what can be associated with carcinogenic toxicants.

Review the description of proto-oncogenes, oncogenes, and oncogene products that occurs in an endocrinology or cell biology text. Note that proto-oncogenes are normal genes, they are not strictly associated with transformation. Oncogenes tend to be normals genes that are inappropriately expressed with respect to developmental timing or level. They may be, but do not have to be, altered structurally. In fact, many are structurally normal, but are altered in genetic position or in their regulatory domains so that they are inappropriately expressed. Those that have been described often include elements of transmembrane signalling pathways associated with cellular growth and division. Note the proto-oncogenes or the oncogenes themselves may be quite neutral in cellular transformation, what is important is the inappropriate expression and subsequent actions of the oncogene products. If chemicals cause alterations in DNA synthesis or repair that result in mutations to proto-oncogenes or their regulatory regions, the result is the oncogenic expression of the altered proto-oncogene (now referred to as an oncogene). The only other route to this condition is a toxicant alteration of the expression of a particular proto-oncogene (or a set of such genes) so that it acts as an oncogene. This might be via a targeted mutation or a specific alteration in the biochemical pathways leading to expression (or suppression) of that particular gene product.

Note that the above view of the integration of proto-oncogenes and oncogenes into cellular physiology seems to differ from the picture of the "foreign invader" provided in the text. It views cellular transformation as a natural process exascerbated by the presence of toxicants, not as a novel process imposed on the system by the presentation of a toxicant.

File counteretalPNAS98.pdf (447 KB) Hide the file counteretalPNAS98.pdf. Edit information about the file counteretalPNAS98.pdf Delete the file counteretalPNAS98.pdf. File dangerturn.shtml (14.7 KB) Hide the file dangerturn.shtml. Edit information about the file dangerturn.shtml Delete the file dangerturn.shtml. File jacks&weinbergscience98.html (51.3 KB) Hide the file jacks&weinbergscience98.html. Edit information about the file jacks&weinbergscience98.html Delete the file jacks&weinbergscience98.html. File Lees&WeinbergPNAS99.pdf (46.4 KB) Hide the file Lees&WeinbergPNAS99.pdf. Edit information about the file Lees&WeinbergPNAS99.pdf Delete the file Lees&WeinbergPNAS99.pdf. File lundber_weinberg98.html (165 KB) Hide the file lundber_weinberg98.html. Edit information about the file lundber_weinberg98.html Delete the file lundber_weinberg98.html. File orrweaver&weinberg98.pdf (223 KB) Hide the file orrweaver&weinberg98.pdf. Edit information about the file orrweaver&weinberg98.pdf Delete the file orrweaver&weinberg98.pdf. File polly.html (28.8 KB) Hide the file polly.html. Edit information about the file polly.html Delete the file polly.html. File weinbergNature.pdf (247 KB) Hide the file weinbergNature.pdf. Edit information about the file weinbergNature.pdf Delete the file weinbergNature.pdf. Close the folder Session 15. Genetic Toxicology: Beginning Coverage of Chronic Toxicity Testing. Folder Session 15. Genetic Toxicology: Beginning Coverage of Chronic Toxicity Testing Hide the lecture Session 15. Genetic Toxicology: Beginning Coverage of Chronic Toxicity Testing. Edit information about the file Session 15. Genetic Toxicology: Beginning Coverage of Chronic Toxicity Testing Permanently delete the lecture Session 15. Genetic Toxicology: Beginning Coverage of Chronic Toxicity Testing and all of its contents. Notes:

Session 15.

Comments on Prometheus -- Where are messages going as they are not appearing as e-mail?

Projects -- appear to be progressing well.

Genetic Toxicology: What is covered?

1. Anything that changes DNA [Joanne]

2. Others?

It includes discussions of:

1. some forms of neoplasia/cancer

2. inherited disorders

3. teratology (gestational losses)

4. ?somatic mutations (such as those responsible for immune system variation)

What are changes in DNA or mutations?

mutations leading to truncations

1. missense

2. deletions or insertions

1. point mutations

a. transitions (G<->A, C<->T)

Because wobble in the third base of many amino acid t-RNA codons allows transitions, transitions will often produce silent mutations.

b. transversions (G<->C, A<->T, G<->T, A<->C)

Because wobble in the third base of many amino acid t-RNA codons often allows transitions but not transversions, transversions are more likely to yield a nonsilent or missense change that will either change an amino acid in protein products of translation or will produce an inappropriate stop codon and a truncated gene product.

2. larger changes

a. frameshifts (1 base deletions or insertions)

These frequently arise during dimer or adduct formation and repair. They may give rise to truncations or gene fusions.

b. large deletions (often occur in repetitive sequences during synapsis in meiosis)

c. sequence duplications (often occurring in repetitive sequences during synapsis in meiosis)

d. inversions (often occur in repetitive or palindromic sequences during synapsis in meiosis)

All of the larger changes can give rise to more obvious chromosomal changes including: insertions, deletions, duplications, inversions, and translocations.

Additional chromosomal changes are possible due to problems arising at the cellular level during meiotic or mitotic division. Chromosome breakage or nondisjunctions fall in this class and often give rise to trisomies or aneuploidies. Note the mechanisms by which these may arise do not have to target DNA, they may come about by interference with the proteins and metabolic pathways responsible for the mechanics or regulation of cell division. They include spindle proteins, centrosomal proteins, centromeric proteins, telomeric proteins, aster proteins, cytokinesis proteins, nuclear membrane proteins and others.

Germline mutations

1. nondeleterious?: recessive, silent, and conservative amino acid substitutions

2. deleterious: chromosomal aberrations, point mutations, deletions, insertions, homozygousity?

Note that most of the conditions we think about are actually the rare cases that are compatible with survival through gestation. They actually represent a minority of the aberrations that actually occur. Losses of zygotes up to the stage of implantation (about 5-7 days postfertilization in humans) is unclear but success in fertilizing mature eggs in vitro is at best 30-50%, with 15-20% being more typical (i.e., losses range from 50-85%). Losses of zygotes and embryoes up to the stage of clear biochemical and clinical recognition (about 2-3 weeks postfertilization) appears to be between 30% and 70% under the best of conditions. Another 15-20% of concepti are lost by miscarriage during the remainder of pregnancy. Thus, somewhere between 24% and 60% of human fertilizations result in a live birth. So 40%-76% are lost due to genetic, metabolic, morphogenetic, or maternal causes. Prior research suggests that genetic causes top the list of reasons for these losses. Thus, evaluating the effects among the survivors may miss the most important elements of genetic deviation induced by exposures to toxicants.

Do they play a role other than in carcinogenesis?

What is teratogenesis?

Is genetic toxicity relevant to the discussion of aging?

Assays for gene mutations.

-- Ames assay

-- Enhanced Ames assay

-- Mammalian cell tests

-- In vivo tests in Drosophila and mice

-- SNP screens

-- microarray evaluations

Assays for chromosome aberrations.

-- Karyotypes of cultured or lymphoid cells

-- Flow cytometry

-- FISH

Other

-- Unscheduled DNA repair synthesis

-- Yeast recombination

-- Sister chromatid exchange

-- Transgenic animals

-- PCR

Prediction of Genetic Insult

Mutagen vs Carcinogen, Chi square evaluation of sensitivity, specificity, positive and negative predictive value

Germ cell vs somatic cell mutagenicity.

Structural cues

Close the folder Session 16. Genetic Toxicology and Testing Methodology. Folder Session 16. Genetic Toxicology and Testing Methodology Hide the lecture Session 16. Genetic Toxicology and Testing Methodology. Edit information about the file Session 16. Genetic Toxicology and Testing Methodology Permanently delete the lecture Session 16. Genetic Toxicology and Testing Methodology and all of its contents. Notes:

Session 16.

Distinctions between carcinogenesis and genetic toxicology?

Degree of gestational losses?

Testing methods

What is the "secret" of the Plains Viscachia? Why might it be a good model for looking at toxic effects on female gametes?

In considering toxicity models for evaluating genetic impacts of toxicants it is worth considering the reproductive strategies being employed by the species being studied. Rodents are a common model yet their reproduction is set up in an R-selected mode. They have a relatively brief lifetime and would not be expected to display a full array of repair, detoxication, or barrier processes that would prevent intoxication of either the adult or developing young. Nor might we expect them to display robust protective mechanisms for gamete production. Many larger mammals including humans, on the other hand, are K-selected and would be expected to display a full array of protections for gametes, developing offspring, and adults. Is there any good evidence for or against these statements?

R-selected: life history favors early reproduction, high rates of reproduction, usually small offspring requiring minimal parental investment, quantity vs quality, favored in a variable environment

vs

K-selected: life history favors late reproduction, low rates of reproduction, large offspring requiring much parental investment, quality vs quantity, favored in stable environments or in species with large ranges

Why might the DAZ1 gene make a good biomarker for testing genetic toxicity in male gamete production?

In humans DAZ1 (Deleted in Azoospermia), resides on the distal portion of the euchromatic portion (the part that is transcriptionally active and has been sequenced and mapped; for the Y chromosome the map is a physical one because only the distal part of the short arm, the psuedoautosomal region, can undergo crossing over during meiosis I when it pairs with the homologous region of the long arm of the X chromosome) of the long arm (q, not for quinacrine, an antimalarial and a green fluorescent stain for AT rich DNA, but for the next letter after p which stands for, petite, in reference to the short arm of a chromosome; quinacrine is a common stain for generating band patterns that assist in karyotyping) of the Y chromosome, just proximal (relative to the centromere) of the heterochromatic region (the part that is not transcriptionally active and is highly repetitive) of the long arm of Y. It is a gene identified by David Page et al. at the Whitehead Institute at MIT as part of a cluster of duplicated genes that is found to be lost in a high percentage (2-20%) of men clinically demonstrated to be azoospermic and therefore infertile. About 15% of couples are infertile in western societies. About 50% of infertile couples demonstrate male-associated infertility including ~35% among them demonstrating male azoospermia. Thus, about (0.15 x 0.1 x 0.35 = 0.00525 = 0.525% or 1/190) 1 in every 200 males lack part or all of the DAZ1 gene!

Because DAZ1 and its clustered homologs are repeats and contain repetitive sequences within each gene, it is not very surprising to find high rates of deletion occurring during the meiotic process involved in spermatogenesis. Since the deletion is apparently nonlethal for the initial stages of development, it does not compromise the vitality of the males that harbor the gene – it simply makes them unable to reproduce. Clearly each DAZ1 deletion that is found represents a de novo mutation occurring during spermatogenesis in the father of the affected offspring. (The gene product of DAZ1 appears to be an RNA binding protein that affects progression of spermatogenesis beyond the spermatocyte stage. It may block meiosis II.)

The high endogenous rate of deletion for DAZ means the site is labile. This makes it a potentially useful marker for monitoring genetic toxicant insults to male gamete production, particularly if the homologs in other species besides man and the great apes also lie in the region in which they are found in man.

Background on genetics of gametogenesis and DAZ, in particular, are found at the following sites:

Lecture on chromosomal biology:

http://www.bi.umist.ac.uk/users/mjfasjw/2MMB/chromosomes/lecture1.asp

Description of plant and animal chemicals:

http://waynesword.palomar.edu/chemid2.htm

DAZ1 technical gene description:

http://gdb.tokyo.jst.go.jp/HOWDY/HOWDY.pl

DAZAP Article:

http://www.biomedcentral.com/content/pdf/1471-2164-2-6.pdf

DAZ circa 1998:

http://wwwvet.murdoch.edu.au/spermatology/iss982.html

Frequency of azoospermia/AZF deletions:

http://www.ich.ucl.ac.uk/cmgs/ydels99.htm

European Guidelines on Diagnosis of Y Chromosomal Deletions:

http://www.emqn.org/guidelines/azf.htm

Male infertility, genetic analysis of the DAZ genes on the human Y chromosome and genetic analysis of DNA repair.

Fox MS, Reijo Pera RA. Departments of Obstetrics, Gynecology, and Reproductive Sciences, Physiology and Urology, and Programs in Human Genetics and Cancer Genetics, University of California, 94143-0546, San Francisco, CA, USA

Many genes that are required for fertility have been identified in model organisms (). Mutations in these genes cause infertility due to defects in development of the germ cell lineage, but the organism is otherwise healthy. Although human reproduction is undoubtedly as complex as that of other organisms, very few fertility loci have been mapped (). This is in spite of the prevalence of human infertility, the lack of effective treatments to remedy germ cell defects, and the cost to couples and society of assisted reproductive techniques. Fifteen percent of couples are infertile and half of all cases can be traced to the male partner. Aside from defects in sperm production, most infertile men are otherwise healthy. This review is divided into two distinct parts to discuss work that: (i) led to the identification of several genes on the Y chromosome that likely function in sperm production; and (ii) implicates DNA repair in male infertility via increased frequency of mutations in DNA from men with meiotic arrest.

Mol Cell Endocrinol 2001 Nov 26;184(1-2):41-9

Source: www.ncbi.nlm.nih.gov

The AZFc region of the Y chromosome features massive palindromes and uniform recurrent deletions in infertile men.

Kuroda-Kawaguchi T, Skaletsky H, Brown LG, Minx PJ, Cordum HS, Waterston RH, Wilson RK, Silber S, Oates R, Rozen S, Page DC. Howard Hughes Medical Institute, Whitehead Institute, and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Deletions of the AZFc (azoospermia factor c) region of the Y chromosome are the most common known cause of spermatogenic failure. We determined the complete nucleotide sequence of AZFc by identifying and distinguishing between near-identical amplicons (massive repeat units) using an iterative mapping-sequencing process. A complex of three palindromes, the largest spanning 3 Mb with 99.97% identity between its arms, encompasses the AZFc region. The palindromes are constructed from six distinct families of amplicons, with unit lengths of 115-678 kb, and may have resulted from tandem duplication and inversion during primate evolution. The palindromic complex contains 11 families of transcription units, all expressed in testis. Deletions of AZFc that cause infertility are remarkably uniform, spanning a 3.5-Mb segment and bounded by 229-kb direct repeats that probably served as substrates for homologous recombination.

Nat Genet 2001 Nov;29(3):279-86

Source: www.nature.com

Gametogenesis in diagrams:

http://gened.emc.maricopa.edu/bio/bio181/BIOBK/BioBookmeiosis.html

http://www.tjc.edu/science/images/reproduction/female_anatomy.htm

http://www.nature.com/nrg/journal/v1/n1/slideshow/nrg1000_040a_F2.html

http://www.sunydutchess.edu/ahbs/Faculty/Scala/Bio102/PDF/Spermatogenesis.jpg

http://www.tyler.cc.tx.us/science/images/reproduction/male_anatomy.htm

http://www.cvm.okstate.edu/instruction/mm_curr/histology/MR/himrp4.htm

Note the efficiencies of gamete formation in mammals where there is physical competition among sperm for ferilization vs the "chemical/hormonal" competition that occurs during the maturational phase of oocytes.

Note also there are important differences in somatic versus germ cell mutations and how they are evaluated.

Brief history of pharmaceutical industry 1900s:

http://www.aphanet.org/about/sesquisept01.html

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Session 17. Assays

Discussion on merits of form of final: take home, in class/ open book, in class/ closed book? A hybrid is probably best, part take home, part in class objective.

In running assays how do we account for alternative causes and background? This requires being able to distinguish a signal due to the causative agent from a signal due to background noise. This is best accomplished by use of an assay that is well characterized and validated. For both bioassays or analytical assays this means the assay must operate within well-characterized ranges for a series of quality control parameters. These are criteria for valid assays:

1. Precision -- reproducibility (A given input results in a predictable output.)

2. Accuracy -- evaluation of reference standards yields expected results (as determined by an independent method or from a consensus of others running similar tests on the same standard materials)

3. Specificity -- measures the intended analyte or parameter even in the presence of potentially interfering substances (lack of specificity = nonspecificity or cross-reactivity)

4. Sensitivity (Limit of Detection) -- lowest measurable, nonzero dose (e.g., LOAEL); also, in a chemical analytical sense, the slope of the response curve, dY/dX, the ability to differentiate one dose from the next.

Bias is found when a result curve is obtained that is different from that seen in another assay. The idea combines sensitivity and specificity dimensions.

Note that a high signal to noise ration, S/N, implies a high analytical sensitivity. It will do well in discriminating the presence of signal from background.

Comparisons among assays are often evaluated using a Chi-square approach where the distinct possible outcomes of the reference assay or evaluation (for example, development of disease or the known content of a compound) are placed on the Y-axis and compared to the outcomes of the test assay (or drug trial). For a 2x2 evaluation where each test has yes or no answers (i.e., they are qualitative or semiquantitative) each of the cells can be readily described.

1. When both tests give positive assays, the result is entered in a square termed "true positives," TP.

2. When both are negative, the result is entered in a square termed "true negatives," TN.

3. When the reference test is positive, but the test assay is negative, the results are "false negatives," FN.

4. When the reference test is negative, but the test assay is positive, the results are "false positives," FP.

With this information, the test assay can be evaluated (relative to the reference) by five qualitative features:

1. Sensitivity = (TP/(TP + FN)); note, this is not the same as lowest measurable dose or slope of the curve for a single assay.

2. Specificity = (TN/(TN + FP)); this is related to, but not the same as measuring the intended parameter.

3. Predictive Value for a Positive Test = (TP/(TP + FP))

4. Predictive Value for a Negative Test = (TN/(TN + FN))

5. Efficiency = ((TP + TN)/(TP + TN + FP + FN)); this is the overall ability of the test to correctly predict or of a compound or drug to have the expected result.

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Session 18. Developmental Toxicology 1

The final needs to be out by May 15.

Genetic vs Developmental Toxicity

Genetic toxicology deals with somatic (could include carcinogenesis), and germ line toxicity.

Developmental toxicology deals with defects arising from problems of gene transcription and expression, cellular biochemistry, intercellular signalling (epigenetic problems); and, it deals with these during embryology, gestation, early growth, and maturation.

Developmental toxicology is often held synonymous with teratology (the science of abnormal development, including biochemistry, morphogenesis, and behavior). Classic examples are phocomelia (flippered limbs resulting from maternal exposure to thalidomide) or cyclopia (a highly visible midline developmental defect occasionally encountered in obstetrics).

Note that much of development is governed by the products of the HOX genes which initiate elements of developmental differentiation programs.

Examples include those genes determining bilateral symmetry, body segmentation (in insects, e.g.), specific organ development (e.g., SRY, SOX3, SOX9, in gonad differentiation).

Intrinisic genetic programs and intercellular signalling patterns ("induction gradients" e.g., as studied in the eggs of developing amphibians like frogs and salamanders -- amphioxus-- where small segments from a morula, blastula, gastrula, or neurula when transplanted from one site on the structure to another site give rise to the normal outcome of the first site, e.g., extra eyes or legs).

The stages of development in the mammal are summarized briefly as:

1. Fertilized egg (found near mid-cycle at the time of conception, prior to sperm and oocycte pronuclei fusing)

2. Zygote (one cell embryo)

3. Morula ("ball of grapes," up to about 32 cells; stage at or just before entry of the early embryo into the uterus)

4. Blastula ("hollow sphere," up to about 200 cells; stage at which blastomeres begin to speciallize, about 90% go to form the trophoblastic cells that presage the pregnancy membranes and placenta, the supportive tissues; these are the earliest source of hCG beginning about day 7-10 in the human; about 10% of the cells are now embryoblasts that form the inner cell mass and will make up the embryo proper as well as portions of the amnion and yolk sac). About day 3-5 post ovulation the blastula enters the uterine cavity and soon breaks free of the zona pellucida that covers all stages prior to this one during their journey from the ovary through the Fallopian tube. About day 5-7 the blastula begins the process of nidation or implantation by interacting with the deciduallized portions of the endometrial lining of the uterus.

5. Gastrula (This stage follows during the period of implantation. It is the period during which distinct germinal tissue layers differentiate to produce distinct endodermal, mesodermal, and ectodermal cell layers within the embryo.)

6. Neurula (Immediately following gastrulation the neurula forms as the early stages of the notochord and neural development take place along with muscle and somite development.)

7. Embryogenesis (The embryo proper arises during this period of organogenesis.

8. Fetal Development (When the embryo has formed the tissues and organs most of the job of development is growth and maturation. This is taking place during the fetal period of gestation.)

9. Neonatal Development (Following parturition or birth, rapid growth and maturation of structures continues immediately following birth and up to about the third year in humans.)

10. Childhood Development (Slower growth and maturation of most structures except the brain, which continues to grow and develop rapidly, marks this phase of organismal development.)

11. Puberty (Hormonally triggered changes result in sexual and metabolic maturation of many systems.)

12. Adulthood (Also known as the reproductive period. Growth slows.)

13. Aging/Senescence (Gradual decline in functionality, growth becomes negative.)

To track progress through these various stages a number of useful biomarkers have been identified. Ultrasound can allow monitoring of the growth and rupture of ovarian Graafian follicles, implying the shedding of a mature egg. It can also be used to monitor the uterine endometrium for the development of an implantation site (at about 3-4 weeks post ovulation in humans; it provides an alternative to palpation for clinical diagnosis of a pregnancy). HCG is unique to pregnancy and neoplasias and can be monitored in blood or urine beginning about 7-10 days postovulation; present in apes, it is absence in many other species largely due to the variations in uterine-embryo associations during gestation and the dependency of the pregnancy on the function of the corpus luteum in early pregnancy. Blood or urine metabolites of the major estrogen and progestins arising from the ovary (and later the placenta) are more variable than hCG and must be tracked longitudinally, but they will remain elevated beyond their normal 10-12 days post ovulation if a pregnancy becomes established (in a nonfertile cycle they decline rapidly near the end of the cycle). This would also be the timing of the first missed menstrual period in humans.

Under normal conditions, most of the fertilized oocytes and early embryoes are lost, easily >50%. Yet toxicants can elevate even this level or can cause defects in particular organ systems by interfering with the normal coordination and regulation of the developmental program.

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Each of the projects submitted under Files/Collaborative in response to the instructions for the projects was examined. Several have problems with preservation of links generated during database searches. These documents may need to be downloaded initially as files and then transferred as supplementary files linked to the master via hyperlinks. Incorporation of hyperlinks to references may be accomplished either by inclusion of the links in footnotes, or by common hyperlink references for the footnote and the bibliographic citation. Note that the wording on the presentation page does not need to match the text in the hyperlink.

Of more toxicological, rather than technical, substance is the need to address each project authors ideas about synthesis and integration of the information gathered regarding compounds or case studies with respect to use of toxicological approaches, risk assessment, and whether any resolution of the problems involved were actually found. This needs to appear as part of the composition. Thus, though the HTML document may include some lists or tables, it should go beyond this to make significant comments about the information that has been gathered. There needs to be an analysis done and presented that covers the chosen project topic.

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Session 20. Developmental Toxicology II

Subsequent sessions will cover Reproductive Toxicity (Definition of reproductive toxicity, Forms of reproductive toxicity), Environmental endocrine disruptors, Stress and dose interactions, Dietary interactions, Remedial actions, Summation

ASTM testing methods:

chronic oral toxicity: http://www.astm.org/DATABASE.CART/PAGES/E1619.htm

ASTM Bioassay Table:

http://www.cwo.com/~herd1/bioassay/Table.htm

How would one go about testing whether a compound was a developmental (versus a genetic) toxin?

One suggestion offered was to begin by checking the components, such as enzymes, in fetal circulation. This, however, raises the question: "How do you sample fetal blood?"

To consider this it is helpful to look at the anatomy of several stages of fetal development.

Once in this site go to Biology 30, Unit 2, Human Development:

http://www.telusplanet.net/public/firstaid/biology30/develop.htm

The Visible Embryo, informational site at NIH: http://www.visembryo.com/

University of Vermont teaching modules for human embryology:

http://cats.med.uvm.edu/cats_teachingmod/embryology/cats_embryo_home.html

Ultrasound image galleries for obstetrics and gynecology: http://www.obgyn.net/us/img_gal.htm

Ultrasound image gallery for human development: http://www.obgyn.net/us/gallery/gallery.htm

Gray's Anatomy OnLine: http://www.bartleby.com/107/ illus502.html

Fetal circulation by cardiac catheterization: http://user.gru.net/clawrence/vccl/chpt1/fetcirc.HTM

Gray's Anatomy, Embryology, Placental Development OnLine: http://www.bartleby.com/107/12.html

Note that sampling might potentially occur from the umbilical veins (flowing from the placenta to the embryo) or arteries (flowing from the embryo to the placenta) as early as 4-6 weeks post ovulation. Practically, since the embryo is only 20-30 mm long at 4-6 weeks, fetal blood could probably only be sampled about 8-10 weeks onward once fetal growth is at least 75-100 mm. And that would need to be done with the guidance of ultrasonic imaging. More realistically testing might be accomplished using sampling of chorionic or amniotic fluids or, at very early stages, by sampling of placental villi.

These tests would, however sophisticated, be checks only on the first generation. They might include direct evaluation of enzymes and tissues sampled from placental villi, cells from amniocentesis, both of which yield information on potential genetic toxins as well, from: karyotype; molecular tests like RFLP -- DNA, treated with restriction enzymes like ECORI, gel electrophoresis, and/or Southern blots and probes, or sequencing of bands --, SNP tests using PCR, LCR, or sequencing); PCR; metabolic information from protein and enzyme evaluations (Western blots, 2D gels, enzyme activities, cell growth in vitro); tissue growth information from imaging, e.g., ultrasound or ?MRI; physiology from levels of steroids, proteins, hormones; (maternal) immunoglobulins in fetal compartment(s). They probably would not indicate if development overall was normal or resulted in an offspring capable of optimal function including reproduction.

Indeed, these tests don't really have the capacity to evaluate full development, coordination of tissue growth, or rate of organismal growth until we look at whole animal tests. An acute dose to mom implies an impact or loss of the embryo or fetus and/or a decrease in the capacity to fertilize and implant zygotes. A chronic dose to mom may not lead to as precipitate a response. Either might allow testing of early stages as above. Lower, chronic doses may also allow examination of developmental imacts on the F1 generation either as assessed directly by the kinds of tests listed above or more overall impacts on growth and development as assessed up to and after birth and through reproduction of the F1 to produce F2 progeny.

Developmental toxins may not necessarily change the genetics of the F1 generation, they are distinguished by postgenetic impacts as often seen in changes in cell structure and/or physiology. Sometimes thes are readily apparent, sometimes they are so subtle they are not apparent until the F1 is functioning outside the uterus or even attempting to produce the F2 generation.

Multigeneration tests imply the need to use small animals to allow handling of enough individuals to statistically see the increment of alterations caused by the toxicant above those occurring normally (often at rather high rates, e.g., in cases of loss of early embryos). The numbers need to allow estimations above a high level of natural background "noise." These tests also require the use of animals with a short generation time so enough tests can be run in a short period of time to allow many compounds to be tested in a reasonable period. Mice are obvious candidates that fit these needs.

But notice that use of such short lived animals may run risks of missing important problems that arise only in longer lived species that require other or more coordination of developmental events over a more prolonged lifespan, e.g., maintenance of stem cell lineages. This is also complicated by differences in the levels and types of protection/repair/detoxification processes occurring in small versus larger longer lived species.

Discussion also centered on some of the special sensitivities that arise in the developing embryo or fetus:

1. Organs may function differently in the fetus versus the adult, e.g., the liver is hematopoietic in the fetus, the adrenal cortex is hyperplastic and very active in a modification of steroidogenesis peculiar to the fetus, blood flow in the heart is different.

2. Key molecules in the adult differ from those in the fetus, e.g., fetal hemoglobin has a higher affinity for oxygen than adult hemoglobin implying it also has higher affinities for CO, CN, and NO and therefore potentially higher susceptibility to such gases or products of endogenous pathways in mother or fetus producing such compounds.

3. The placenta itself is a site of high P450 enzyme activity in relation to steroidogenesis and detoxification/toxin activation.

4. Rapidly growing fetal tissues are sites of high expression of receptors for growth factors and of paracrine growth factors themselves. They rapidly take up molecules involved in macromolecular synthesis and overall metabolism. Therefore, they have increased susceptibility to structural mimics of such building blocks and/or inhibitors of growth processes, e.g., lead vs calcium, phorbol esters vs diacyl glycerides, D- vs L- amino acids, L- vs D- glucose.

5. Unique activities are occurring in the embryo versus the adult, e.g., active growth of nervous and eye lens tissues, migration of germ line cells, migration of various cell lines (Rathke's pouch> anterior pituitary, ultimobrachial anlage> thyroid and parathyroid tissues). Some signalling paths may be unique to the embryo or fetus, e.g., chemotactic substances, certain transcription factors.

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The next sessions will cover: Endocrine Disruption, Dietary Impacts on Toxicity (Food Toxicology), Stress & Toxicity, Remediation Methods, and a final Overview

For Endocrine Disruption next week: Bring in one article from the popular press and be ready to discuss it.

This session covered the physiology of the reproductive systems of the male and female including the multiple levels of intercellular communication necessary for normal function to occur. Note that this provides many targets for potential disruption by pharmacological or environmental agents. Regulation by the neuroendocrine components of the hypothalamus provide a target for a variety of neurotoxic agents that may ultimately be expressed as reproductive disruption and, therefore, toxicity. Secondary controls on hypothalamic control such as the stress axis (adrenal axis) and leptin regulation provide additional areas that may be disrupted to external agents yet express themselves as reproductive problems. Disruption of the feedback regulation exerted by gonadal steroids on the hypothalamus and hypophysis provide yet another target for disruption of reproduction at the level of the brain and pituitary. But potential targets also exist with respect to the binding and actions of the gonadotropins at their primary target cells in the gonads, the cells supporting gamete production.

Leydig cells in the testis and granulosa, thecal, stromal, and luteal cells in the ovary are all steroidogenic. They make use of cholesterol and cholesterol ester stores sequestered as lipid droplets to generate the gonadal steroids that both support gonadal and reproductive tract function and provide the systemic feedback to regulate hypothalamic and hypophysial control of reproduction. Thus, these cells attract and concentrate lipophilic compounds that are capable of physically partitioning into fat. Brain tissue also tends to be vulnerable in this way because of the concentration of lipids in myelin. Oocytes themselves tend to accumulate such materials because of their yolk content. Sperm are spared due to their comparatively low lipid content.

The Sertoli cells and male gametes are vulnerable to other agents. Those capable of disrupting tight junction formation may break down the junctional complexes between Sertoli cells and developing spermatogenic cells, disrupting the blood testis barrier that is needed to maintain integrity of the semeniferous tubule compartments and keep spermatogenesis well orchestrated. In contrast, the granulosa cells (especially of the cumulus layer) and the oocyte are vulnerable to agents that disrupt gap junctional complexes since these are needed to coordinate the functions of the ovarian follicle and the growth of the oocyte.

While the spermatogenic cells are particularly sensitive to toxins affecting rapidly growing cells, mammalian oocytes are sensitive to agents that interfere with both meiosis and apoptosis (the normal manner in which oocytes begin their decline toward atresia). Agents that speed the loss of oocytes can lead to premature depletion of the oocyte pool because that pool is physiologically limited in size at mammalian birth. Spermatogenesis on the other hand seems relatively robust but can still be disrupted by any agent capable of modifying the sequence of events leading to formation of normal spermatozoa or their transport to the female tract.

The female tract itself is a sensitive target because of the cyclical proliferation, differentiation, and regression that takes place in the tissues of the endometrium, and, to a lesser extent, in the vagina. Steroid mimics, agents that disrupt cellular proliferation (or regression), or those that alter paracrine signal transmission among the tissues of the female tract can change the timing or movement of gametes and/or zygotes and blastulae through the female system. Changes in cilliary beat in the Fallopian tube, changes in fimbrial activity, alterations in cervical mucus secretion or biochemistry, alterations of female tract contractility or contractile responses to seminal prostaglandins can all affect the success of gamete movement, fertilization, and/or implantation and gestation.

Finally, any agents that disrupt enterohepatic metabolism of steroids or other reproductive tract signalling agents will have potential reproductive toxicity effects. Either they will cause steroids to be cleared too rapidly and therefore will alter the normal conversation between the gonads and the hypothalamus/pitutitary or they will cause steroids to be cleared too slowly, similarly disrupting this set of feedback equilibria.

In testing for such impacts we again run into the problem seen with genetic and developmental toxicity. To determine if the toxin is effecting the parents, offspring must be generated. But because the offspring may themselves be carriers of the toxic defect imposed by exposure of the parents to the toxic agent, the offspring must be evaluated as well as the parents for anomalies of structure and function. And function includes reproductive function. Thus, reproductive toxicity tests, as they exist at the present time, include evaluation through three generations (parental, F1, and F2) to ascertain what the impacts on reproduction might be. Close the folder Session 22: Endocrine Disruption. Folder Session 22: Endocrine Disruption Hide the lecture Session 22: Endocrine Disruption. Edit information about the file Session 22: Endocrine Disruption Permanently delete the lecture Session 22: Endocrine Disruption and all of its contents. Notes:

Session 22: Endocrine Disruption

Discussion of the articles brought in.

Allison: An article concerning disruption of sexual development in birds on the West coast as a result of exposure to chlorinated hydrocarbons.

Joanne: An article from the Ecologist regarding possible lindane contamination of chocolate from Ghana as a result of economically driven agricultural practices.

Neetu: A Science News article describing the effects of arsenic as an endocrine disruptor.

The first two articles remind us that the legacy of synthetic polychlorinated hydrocarbons is still with us. Even 40 years after the publication of Silent Spring and the governmental actions in setting up the EPA, NIEHS, and strengthening the FDA, these pesticides and their relatives remain a persistant problem. The impact of chlorinated aromatics including DDT and PCBs has continued to unfold since their development in the 1930s and their association with the decline of predatory bird species described in the 1960s and early 1970s. Our own Dr. Jeremy Hatch works on a population of roseate terns who have a decreased fertility and marked decrease in the proportion of males due to contamination of food sources near Cape Cod with chlorinated hydrocarbons. The Wisconsin Raptor Center and the WARF Institute played a major role in describing the decline of predatory bird species as a result of egg-shell thinning, calcium metabolism problems, and vitality reduction associated with accumulation of excessive levels of these compounds in bird fat and egg yolk. The compounds accumulate up the food chain, concentrate in fatty tissues, inhibit the carbonic anhydrase necessary for egg shell formation, and tend to be "off-loaded" in the fatty yolk of the eggs. Thus, young are exposed to even higher levels than are adults during crucial times of development. Although PCBs were in use when DDT was being identified with bird population problems, it wasn't until the mid- to late 1970s when it became fully apparent that this series of widely used electrically nonconductive, fire retardant, heat dispersing agents was leaking into the environment in significant quantities and generating many of the same kinds of problems the chlorinated hydrocarbon pesticides had.

All of these compounds are resistant to degradation but gradually give way to the actions of mixed function oxidases and/or exposure to intense UV irradiation or to high heat. They are slowly oxidized to related ketones, carboxylic acids, and alcohols that are themselves slowly broken down by P450 type enzymes and/or conjugated to glucuronic acid, sulfate, or amino acids to yield more water soluble compounds. Along the way, however, these compounds and some of their metabolites may mimic estrogens or thyroid hormones.

The issue of arsenic as an endocrine disrupting agent is somewhat puzzeling, however, arsenic's position in the periodic table suggests it may interact and/or interfere with metabolic processes involving phosphorylation. As this is common during many transduction processes including the movement of glucocorticoid receptors to the nucleus with the concommitant dissociation of a series of heat shock proteins, it may not be so surprising that arsenic may have some specificity in its actions in such systems. It may also be possible that arsenate's resemblance to vanadate plays a role because vanadate is commonly used during in vitro experimentation to stablize isolated glucocorticoid receptors. Such stabilization reduces hormone-receptor turnover and may lock the expression of certain genes either "on" or "off."

As a topic of active interest currently and in the recent past there are many references that can be pointed to. The reading list contains at least five book titles addressing this topic besides the text.

Although most attention has been directed to the issue of environmental estrogens and/or environmental anti-androgens, thought should also be given to compounds that have potential to disrupt the thyroid axis, vitamin D metabolism, retinoid growth regulation, and the adrenal axis.

Coverage in C&D pays particular attention to tumorigenesis and induction of neoplasias in the endocrine tissues. It also notes, however, that many of these effects are the indirect result of disruptions of normal homeostatic feedback loops with much of the hyperplastic induction being due to chronic elevations in trophic hormone production and target tissue stimulation. While impacts that produce neoplasias are definitely dramatic, they are really the extreme manifestations of functionally more important disruptions of endocrine homeostasis. Considerable note was taken of the apparently higher susceptibility of rodents, particularly some of the favored toxicity testing strains such as Fischer 344 rats, to formation of endocrine tumors relative to what is seen in other rodent strains, or other species including humans. Is it worth considering how functional tests of the endocrine axis might serve as more sensitive biomarkers for toxic insults to these systems than waiting for rats to develop visible tumors?

Note that many of the considerations we mentioned in discussing reproductive toxicity again arise when examining the overall endocrine axes: lipid uptake by steroidogenic tissues, actions of steroidogenic P450 enzymes on xenobiotics to produce proximal toxicants, interferences with enterohepatic breakdown of lipoid hormones causing disruptions of feedback loops, and modification of the molecular loads carried by hormone binding globulins in circulation.

There are few big surprises here, mainly logical outcomes of the physical chemical and physiological characteristics that make up the endocrine system itself.

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Components of food as additives

Unintentional (regulated or incidental)

Intentional (regulated or GRAS)

Constituents (unregulated)

GRAS = generally recognized as safe

Traditional (safe by experience -- human epidemiological evidence)

Safe by testing

Focus on potential carcinogens is largely due to the Delaney clause written into law in 1958 at the time food safety legislation was being updated to reflect then current scientific and epidemiological evidence.

(http://www.cnie.org/nle/crsreports/pesticides/pest-3.cfm,

http://www.acs.ohio-state.edu/units/cancer/sa97front/Delaney.htm, http://pested.unl.edu/thelabel/tljan98.htm, http://www.rff.org/resources_articles/files/delaney.htm)

The Delaney clause stated that no compounds shown to be cancer causing in animals or humans would be tolerated at a measurable level in food or cosmetics. the legislation, however, allowed those compounds considered GRAS to be used. Note that the "measurable level" at that time was several orders of magnitude higher than what is now attainable. In addition, current toxicological databases include information on a variety of compounds originally on the GRAS list (such as food colors) that suggest they may not be suited for chronic, high dose use in foodstuffs. Note also that the Delaney clause has been interpreted to apply only to induction of primary carcinogenic lesions, it does not cover induction of a state leading secondarily to neoplastic lesions as might be the case for an endocrine disrupting chemical.

Common food additives include at least 30 categories of compounds including:

Preservatives (e.g., MSG, BHT, BHA)

Nutriative Supplements (e.g., Vitamins A, C, D, E, K; iodine)

Antioxidants (e.g., Sulfites, EDTA, PABA, vitamins A, C, E)

Nonnutriative Supplements (e.g., oils, sweeteners)

Fillers

Flavors

Colors

Scents

Processing agents

Note that colors, flavors, and scents often contain aromatic, or polyaromatic compounds that tend to be lipophilic. In the case of colors, they are often potential DNA intercalators and therefore DNA synthesis disruptors that may lead to induction of neoplasia.

Food contaminants include:

Pesticides

Packaging constituents

Metals

Animal drugs

Food toxins (of plant, animal, or mycotic origin)

Bacterial (including pathogens that are virulently infectious & those that are highly enterotoxic)

Maturation of the gut plays a major role in determining just how problematic some of these contaminants may be. Until maturation allows full acidification of the stomach and maturation of the blood-lumen barrier in the gut, a variety of toxins that are normally acid-labile and/or macromolecular can access portions of the digestive tract where they can be absorbed and taken into general circulation. This may allow action by acid-labile enterotoxins, e.g., botulinum toxin, establishment of inappropriate antigenic tolerance (anergy), and/or alteration of host/pathogen interactions that may range from deleterious to beneficial.

Not only do contaminants in food modify the consumer, constituents in food may modify the consumer's exposure to and metabolism of certain toxicants.

Fiber and lipids may alter gut transit time, the release of toxicants to the consumer, the enterohepatic cycling of the toxicant and its metabolites to and from the gut contents, and the elimination of the intact or metabolized toxicant from the consumer. These constitutents may modify the kinetics of toxicant entry into the organism or its metabolism of these toxicants.

Modulation of stomach acidity by ingestion of a vegetable or high protein diet may also change the dynamics of toxicant entry into the consumer.

Food content of antioxidants such as vitamins A, C, and E may directly modulate metabolism in the gut or alter the redox status of tissues like the liver involved with toxicant catabolism. This would include any food components that stimulate phase I or II enzymes; compounds with hormonal activities that impact hepatic or gut function (steroids, neurotransmitters or precursors); or, compounds with pharmacologic activities that also impact catabolic tissues (caffeine, salycylic acid, nitrates). Close the folder Session 24: Stress and Toxicity. Folder Session 24: Stress and Toxicity Hide the lecture Session 24: Stress and Toxicity. Edit information about the file Session 24: Stress and Toxicity Permanently delete the lecture Session 24: Stress and Toxicity and all of its contents. Notes:

Session 24: Stress and Toxicity

Role of heat shock proteins.

( http://ehpnet1.niehs.nih.gov/docs/1998/Suppl-6/1319-1323wijeweera/abstract.html)

Role of adrenal axis.

Role of CNS.

Role of renal blood flow control.

Role of leptin axes.

Types of stress in mammalian cells:

http://www.csa.ru/Inst/gorb_dep/inbios/SSE/2_4type.htm

A primer on Oxidative Stress:

http://www.agronomy.psu.edu/courses/AGRO518/Oxygen.htm

Diagram of pathways:

http://www.sigma-aldrich.com/sigma-aldrich/image/sg_ls_cs_slide30.jpg

Summary reactive oxygen and nitrogen species:

http://www.fedem.org/revista/n8/imagenes/image008.jpg

Hepatotoxicity slide presentation:

http://www.le.ac.uk/pa/msc/heptox.pdf

Thiols in oxidative stress:

http://www.pharmanac.net/pdf_documentation/ OSATDITPAPBLIHIP.pdf

A definition of oxidative stress:

http://www.bb.iastate.edu/~jat/glutchp.html

DNA damage in oxidative stress:

http://www.dojindo.com/newsletter/review_vol2.html

Mitochondrial dysfunction:

http://www.mrc-dunn.cam.ac.uk/research/dysfunction.html

Oxidative stress and calcium:

http://www.grc.nia.nih.gov/branches/lns/linka.htm

A doctor's primer on antioxidants:

http://www.thedoctorslounge.net/education/tutorials/antioxidants/antioxidants1.htm

Oxidative stress in optical systems:

http://www.djo.harvard.edu/meei/OA/lat/INDEX.html

Slide presentation on Vitamin E and oxidative stress:

http://qcom.etsu.edu/nutrition/vit%20e%20presentation/ tsld002.htm

Abstract of a Review:

http://ehpnet1.niehs.nih.gov/docs/1998/ 106p375-384kelly/abstract.html

Abstract of an article on radiation associations:

http://www.lowdose.org/pubs/ehp/docs/klaunigabstract.html

Stress

* Internal stimuli (loss of homeostasis, chemical signal imbalance)

* External stimuli (environmental -- heat, light, pressure, sound, biological interactions between organisms, interpersonal/behavioral)

Note either source of stimulus can be acute or chronic. Stress gives rise to supra- or ab-normal systemic activities. At the cell level these may involve heat shock protein expression, changes in membrane potentials and ion balance, changes in cellular metabolism, changes in gene expression, changes in receptor numbers, etc. At the tissue level, these translate into changes in chemical or hormonal sensitivity, "irritability," capacity to respond or function. At the system level the changes give rise to alterations in excitability and chemical signal output. Examples include:

1. Adrenal axis/adrenal cortex >>> CRH, cortisol, DHEAS levels rise thereby increasing blood glucose levels, enhancing lipolysis, suppressing immune functions among other end results.

2. Adrenal medulla >>> norepinephrine & epinephrine rise thereby altering blood pressure and cardiac rate.

3. Kidney >>> responds to vasopressin by increasing water retention; responds to a fall in renal perfusion by increasing renin output resulting in rises in angiotensin I & II and aldosterone (from the adrenal cortex, glomerulosa layer) with subsequent increases in sodium retention and blood pressure.

4. CNS >>> may increase output of opiate peptides to suppress nociceptive stimuli; may decrease NPY and other peptides while increasing CART, CRH, and other peptides in the leptin pathways resulting in suppression of apetite; may increase output of vasopressin, dopamine, serotonin or other neurotransmitters causing alterations in both sympathetic and parasympathetic nerve transduction pathways.

5. Thyroid >>> may increase output of thyroxine causing generalized increases in metabolic rate and increased thermal output; under chronic stress the reverse may occur; thermal control is compromised under both situations.

6. Immune system >>> interleukins, CRH, bradykinin, and prostaglandins may all be released causing alterations in adrenal, thyroid, CNS and other functions. Note that the kinins and prostaglandins may stimulate smooth muscle contractions of the vascular endothelium or even organ walls -- stomach, intestine, uterus (Clearly infection is an environmental stressor.)

Note several normal biological processes utilize stress as a signal. Disease has been mentioned. Should toxins alter any of the steps involved in these signalling pathways and intracellular signal cascades, they have the potential to either block or exascerbate the stressful condition. The birth process involves stress on both the offspring and the mother. In the offspring near term the maturing thyroid axis and general rapid growth of the fetus brings on a generalized anoxic stress. Generalized "crowding" by the maternal organs including the pelvic bones stimulate the fetal adrenal axis to produce CRH, ACTH, and resultant increased cortisol (and DHEAS). Placental CRH is also rising at this time and helps reinforce this stimulus of the fetal adrenal axis. The adrenal steroids alter the metabolism of steroids and prostaglandins in the placenta. Progesterone production falls in favor of estrogen production while the placental inactivation of prostaglandins that normally occurs during pregnancy is inactivated. The increased estrogens, and decreased progesterone levels favor increases in oxytocin receptor numbers in the maternal myometrial cells. They also increase the maternal systemic stimulation of the posterior pituitary production of oxytocin and the myometrial cells' formation of gap junctions. The oxytocin actions both directly and mediated by formation of prostaglandins in myometrial cells stimulate coordinated contractions of the maternal myometrium and ultimately lead to completion of parturition.

Other systems also contribute to the process: pudendal nerve compression during late pregnancy decreases output of norepinephrine which normally decreases myometrial contractility; opiates are increased dramatically during birth to act as endogenous pain suppressors.

Additional mechanisms may also contribute in this case. For example, progesterone levels which peak at or near term for most mammals may act via glucocorticoid receptors during the latter part of pregnancy to alter glucose metabolism, immune responses, and possibly even help decrease expression of prostaglandin inactivating enzymes, though this latter mechanism has yet to be shown.

Shock is another, if extreme, example of systemic stress that may be accelerated or induced by toxicant actions. Generally breakdown of kidney function or of osmotic tissue barriers leads to decrease in blood pressure, decline in cardiac rate and output, a decrease in oxygen and glucose perfusion of the brain and other organs, CNS depression, respiratory depression, and systemic failure. Any toxicant accelerating this chain of events may cause shock if given in high enough dose.

Currently, the major path for actions of toxicants in causing tissue effects not related to uptake by particular receptors or via indirect impacts on cellular metabolism or cell division, involves the concept of oxidative stress. This is covered in some depth in Hodgson and Smart, Introduction to Biochemical Toxicology, 3rd Ed, 2001, John Wiley & Sons, Inc.: New York, NY, 235-253.

Specific elements of the concept are covered in the links provided at the beginning of this session. The concept entails the idea that cells are damaged as a result of the breakdown in balance of reduction and oxidation within the cells. If oxidative conditions arise as a result of the unchecked intracellular production of peroxide or superoxide radicals, or reactive nitrogen species, these molecules can covalently alter other molecules, including macromolecules, within the cell. Oxidized lipids or proteins will compromise membrane functions so mitochondrial or cell membrane potentials may be altered. Intracellular signalling may also stimulate production of "cell death" signals causing the initiation of the events leading to apoptotic cell death.

The production and redox cycling of glutathione plays a key role in maintaining intracellular oxidative balance. Adequate levels of other reductive species such as retinoids, ascorbic acid, tocopherol, and reduced nicotine adenine dinucleotides help maintain the balance. Overabundance of metals like iron or copper can be problematic. While any toxicant that requires rapid P450 mediated metabolism increases the endogenous risks for overproduction of superoxide and peroxide species that may act as proximal reactive intermediates in causing cellular oxidation or macromolecular modification. Unless the reductase activities and other repair processes can keep up the cell, minimally, and/or the tissue involved may undergo damage from oxidative stress.

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Session 25: Treatment/Prevention of Toxicity

In class final will be May 15 at 10:30 AM.

So how do we go about preventing or treating toxicity?

For the individual:

1. Avoid exposure

2. Minimize dosage

3. Maximize elimination (e.g., modify urine pH, often alkalinize)

4. Block circulation and absorption of toxicant (chelators, antibodies, charcoal, clays)

5. Apply antagonists (for specific molecular targets)

6. Promote repair

(Note that antibodies may be introduced by passive, e.g., antivenins, or active vaccinations.)

For the environment:

1. Modify chemical use and disposal

2. Institute regulations

3. Minimize releases or exposures

4. Isolate polluted/contaminated areas/populations

5. Institute remediation procedures

Note that Education plays a big role in avoiding both individual and environmental exposures as well as in minimizing dosages. Foreknowlege allows comparative risk evaluations to be made. Regulations help codify the knowledge base and provide guidelines for toxicant handling procedures. They externalize comparative risk evaluations. Education also allow formulation of appropriate response information and the description of pertinent treatments to handle detoxication.

What about treatments in the clinical sense?

Procedure usually involves the following steps:

1. Take a complete history of the intoxication (acute, chronic, contributing conditions)

2. Evaluate the subject's condition, identify the poison and dose, if possible

3. From databases or references determine the expected pharmacokinetics for clearance (How long will elimination take and what effects can be expected in the dose range involved?)

4. If the intoxication is acute and recent, act to decrease the dosage.

5. If minimal risks are involved, provide medical support with minimal interventions.

6. If there is a potential for extensive morbidity or mortality undertake active interventions:

o For oral exposure: dilute the stomach contents, pump out the stomach, give one or more cycles of activated charcoal, provide antidotes or antagonists as appropriate.

o For blood exposure: provide chelators or appropriate antibodies, or perform dialysis or transfusion as required.

o Since dermal and respiratory exposures normally pass through the blood the only palliative measures that apply beyond the treatments suggested for blood exposures are removing the subject from the exposure source and doing a thorough washing with appropriate detergents of the exposed dermal surfaces.

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Definition of toxicology, toxicant, toxin.

Types of toxicology.

Modes of exposure.

Meaning and importance of dosage and route of dosage.

Pharmacokinetics.

Catabolism, enterohepatic metabolism.

Methods of measuring acute and chronic toxicity.

Morbidity, mortality, and quality of life indices.

Carcinogenesis, past and current models, testing.

Genetic toxicity, acute and multigeneration testing.

Developmental toxicity.

Reproductive toxicity.

Endocrine toxicity.

Food toxicity, Delaney Clause.

Toxicant interactions with stress.

Modulation of toxicity. Close the folder Session 6. NOAEL, LOAEL, Zero dose; Handling toxins. Folder Session 6. NOAEL, LOAEL, Zero dose; Handling toxins Hide the lecture Session 6. NOAEL, LOAEL, Zero dose; Handling toxins. Edit information about the file Session 6. NOAEL, LOAEL, Zero dose; Handling toxins Permanently delete the lecture Session 6. NOAEL, LOAEL, Zero dose; Handling toxins and all of its contents. Notes: Session 6.

[Discussion leaders notes.]

Approach the following questions:

1. NOAEL, LOAEL. How do these differ from a zero dose? Do they take into account threshholds?

2. How might an organism handle an MTD differently han an LOAEL dose?

Refer to Figure 3-2 of Casarett and Doull.

Distribution

Note the role of speciallized transport systems in facilitating toxicant distribution and the counterpoised impacts of plasma and intracellular/intercellular binding sites for serving as reservoirs decreasing dose while allowing for bioaccumulation and/or prolonging exposure.

Excretion

Processing attempts to increase hydrophilicity to allow renal clearance, hydrophobic routes are inefficient: milk, bile, direct intestinal elimination (Look at the role of insoluble fiber and particulates in the diet.)

Reabsorption/Recycling

Toxication & Detoxication

Reaction of ultimate toxicant with cellular targets.

Chemical reactions: binding and/or reactivity and resulting impact.

Proof of target specificity (see page 41 of Casarett and Doull Companion Handbook):

"To conclusively identify a target molecule as being responsible for toxicity, it should be demonstrated that the ultimate toxicant (1) reacts with the target and adversely affects its function, (2) reaches an effective concentration at the target site, and (3) alters the target in a way that is mechanistically related to the observed toxicity."

Note the number of intercellular communication problems identified as modes of toxicity in subsequent pages describing modes of toxicity.

[Class notes]

Project choices:

Allison: Sweeteners (saccharin, aspartame, acesulfame-K, sucralose, cyclamate)

Neetu: something about radiation/isotopes

Joanne: something about aquatic toxicity?

LOAEL, NOAEL, Zero dose

Note how these appear differently in a sigmoid model of toxicity in which there is no difference between the beginning of the dose-response curve and zero dose, i.e., the response curve is monotonically increasing beginning at zero dose, and in the threshhold model of toxicity in which there is an actual point on the dose axis above zero dose at which the response falls to zero.

In the monotonically increasing case, repeated measuremens of the dose response curve provide an error estimate around the points of the dose curve including the zero dose. The mean of the zero dose + 2 (or 3) standard deviations of the zero dose, define a line through the dose response curve below which a response cannot be distinguished from the impact of a zero dose. Since doses are chosen for testing, any dose falling below the line just described would yield no effect. The highest dose below that line should be the NOAEL dose. The first dose above that line should be the LOAEL. Note the line defined could also be refered to as the maximal tolerated dose, MTD.

The threshhold model is variant of the linear extrapolation model. The latter assumes there is no dose that does not have some effect. If our methods are sensitive enough, this model would, in fact, have no NOAEL or MTD. If, more realistically, there are physiological mechanisms that pose barriers for toxicity, e.g., detoxification systems, sequestration mechanisms, or repair processes, there will be a dose that does not generate a response in the organism. In this situation, there will be no response at a finite, nonzero, dose which can be termed the NOAEL or the MTD. The first dose above that level would be the LOAEL. Operationally, it is normally impossible to distinguish between a true threshhold model and a sigmoid model given the limits of response measurement technologies and the finite number of dosages that can be tested. Thus, the determination of NOAEL, LOAEL, and MTD fall back to the approach outlined above under the sigmoid model.

What happens with delayed effects? Cancer, neurodegeneration, immunocompromise (where opportunistic infections by secondary agents may actually be the ultimate causes of the observed toxic effect). In all these situations the phase delay between exposure and toxic response may be difficult or impossible to detect above background "noise." But the obvious places to begin exploring these situations are in the tissues most potentially affected. In the case of cancer this would probably be most prominent in the tissues demonstrating the most active proliferation: testicular germinal epithelium, bone marrow, lining of the small intestine, placenta, or embryonic tissues. Note how the clearance and repair processes make the connection of cause and effect even harder because the causal agent may be cleared long before the impacts of its effects are expressed by the biological systems triggered to set in motion processes or cascades that are proximally responsible for observed effects.

Practically, these delays have important impacts on the process of testing drugs and food additives since they may take 10-20 years or more to become apparent. Current patents are only good for 17-20 years including much of the time needed for the later phases of testing. There is little wonder companies try to optimize profits through the course of the initial patent as they need to address the costs not only of development, but also of liability litigation that may arise as a result of delayed untoward effects that may not be made apparent during the time of testing. Animal models are not perfect substitutes for humans and not all humans can be adequately modeled even with other humans.

So the question arises as to whether the potential for delayed deleterious toxic effects might be grounds for delaying the granting of a patent, prolongation of the testing phase, governmentalization of the responsibility for prolonged testing, or automatic patent renewal if no such deleterious effects are reported during the initial patent. Close the folder Session 7. How might specific molecular targets be identified for investigation?. Folder Session 7. How might specific molecular targets be identified for investigation? Hide the lecture Session 7. How might specific molecular targets be identified for investigation?. Edit information about the file Session 7. How might specific molecular targets be identified for investigation? Permanently delete the lecture Session 7. How might specific molecular targets be identified for investigation? and all of its contents. Notes:

Session 7.

Project choices:

Allison: good resources appearing for sweeteners as a project.

Neetu: a comparative study of radiation releases at the Savannah River, GA fuel processing facility and the Techa River, Russia, radioactive materials processing plant. [There is a need to focus on only one or two isotopes and/or instances to keep the project manageable.] Plutonium contamination may be one target of the project.

Joanne: ? Leaning toward something to do with developing systems, but need to look at what is in earlier offerings in aquatic toxicology.

Focus on Question 3 of the second set:

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?

Note the molecules needed to synthesize thyroxine, transport it to its somatic targets, activate it in target cells by removing an iodine atom, allow it to activate genes via binding to a receptor sitting on a thyroid recognition element within the DNA, and terminate its actions via oxidation, conjugation and an increased water solubility and urinary elimination. If we see a structural similarity of a compound to a known hormone or physiological compound, the obvious place to look for potential molecular targets are those molecules normally involved in that compound's physiology and actions. In the absence of a structural similarity, but with a known set of end effects, the targets may lie on the paths leading to normal activations of the ultimately affected tissue. As this often involves hormones, hormonal pathways may well be fruitfully explored.

A second example of these principles is provided by the actions of diethylstilbestrol, a potent synthetic estrogen that acts via the same mechanisms as estradiol (but is not buffered by binding to the sex hormone binding globulin that modulates free levels of estradiol). A path similar to that seen with thyroxine works to identify molecular targets of DES.

With respect to the kinds of interferences in normal paths that might be anticipated, these can be classed in the same manner as inhibitors of enzymes are classed:

1. Competitive

2. Noncompetitive

3. Uncompetitive

Competitive inhibitors tend to be molecular mimics that compete with the normal molecule for binding to an enzyme or receptor. Their effectiveness will depend on the relative concentrations of normal ligands as well as the competitor.

Noncompetitive inhibitors ........

Uncompetitive inhibitors ......

All these various classes define types of antagonism, in which a biological function is inhibited. [Agonists on the other hand would promote a particular function. Note that a toxicant can act by being agonistic to the point of damaging the stimulated tissue or organism. Stimulants of the immune system can generate autoimmune responses or anaphylactic response, for example.]

So how might the drugs or toxins involved leave a traceable path within the biological organisms being studied? A good example is provided by drug testing at the Olympics and other international competitions. In the case of small molecules there are often residues of the parent compound or their metabolites that can be found in blood, urine, or saliva for considerable periods of time after ingestion or injection. Part of this is caused by the recirculating nature of degradative pathways and the enterohepatic circulation involved. Part is also due to the natural sequestration that occurs for lipophilic compounds in fat or for other molecules in binding to carrier proteins such as albumin. Applying immunoassays or HPLC, often combined with mass spectral analysis, can allow detection for up to several weeks after exposure for many of the compounds of forensic interest. [Similar testing is done clinically to determine presence of possible cross-reactive drugs within a patient's system, regardless of whether exposure was via legal or illegal routes.]

Protein or peptide drugs may be present as metabolites in urine (or feces). Detection is similar to that for small compounds except that more speciallized mass spectral techniques such as MS/MS (mass spec/mass spec) or MALDI-TOF (matrix-assisted-laser-desorption-ionization, time of flight) which use ionization techniques that allow examination of macromolecules of up to about 20-30,000 Daltons in molecular mass. As many peptides or proteins that are synthetic may include unique amino acids or may be derived from unique clonal gene sequences, they may provide a molecular "signature" that can be tracked analytically.

Failing the ability to track a molecule by the routes above, or in the face of a rapidly acting/rapidly cleared drug, there may still be signs of its actions that are unequivocal. For example, in the case of athletes taking Darbopoietin, a synthetic/recombinant form of erythropoeitin [a kidney hormone that normally stimulates hematopoiesis], it is not the drug that is being tracked initially, but the fact that red blood cell concentrations exceed normal limits (even for the altitude of the locale of the Winter Games, Salt Lake City, about 4000 feeet). Here the physiological signal suggests the presence of the drug which can then be confirmed by more careful analysis of blood or urine possibly following concentration of a biological fluid extract.

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Session 8.

[In responses to questions please include all URLs or references for web sites visited or articles or books cited. Similarly, if examples are cited, make certain their source is noted in your responses.]

Projects:

Allison: continues to find information on sweeteners

Joanne: project will be on some aspect of developmental toxicology/teratology, possibly focusing on water contamination during pregnancy [Note this needs to be narrowed down to one or two compounds or effects in order to make the project doable in the alloted time.]

Neetu: is finding articles on the Techa and Savannah sites, now must focus on one or two isotopes to make certain the topic can be covered.

Toxicology generates its own controls.

Because toxicants often generate effects in tissues or systems that have not been fully characterized previously, toxicity studies must often generate the basic information characterizing the affected system simultaneously with doing the toxicity studies. As a result toxicity studies may proceed more slowly than might be anticipated. However, the result is often a very inciteful picture into both the natural and perturbed biological system. A good example are the studies that explored the impacts on the amygdala of the contaminant, MPP+, in the street drug MPTP that generated a Parkinsonian like condition on young addicts. The insights into how that portion of the brain worked have informed much later work on slow brain viruses as well as the chemistry of neurotransmission. Thus, toxicity studies are important not only for the forensic results or the description of chemical toxicity, but also for the light they shed on normal physiological function.

Repair functions

Repair functions can compensate for some toxicant actions and can complicate the determination of causality for delayed effects of toxins. So what are some of these repair modalities?

They include, among others:

1. compensatory enzymatic pathways

2. new gene transcription

3. initiation/stimulation of degradative paths (for the toxin)

4. induction of apoptosis of intoxicated cells

5. necrotic induction of cellular immune response (limiting toxicant damage)

6. activation of paths for elimination of products of intoxication

7. induction of DNA repair processes

8. wound healing

Although these all play roles, consider the DNA repair processes. Human (and mammalian, since genetic homology across species is >70% and most often >90%) DNA contains ~ 3 billion base pairs per genomic copy. This DNA is not only twisted in a double helix with oppositely oriented strands, it is coiled, and supercoiled. These latter forms of packing make it necessary for cells to use enzymes like helicases or topoisomerases to reverse the coiling and allow DNA replication. However these enzymes must cut the DNA to accomplish their tasks. As a result, the replicating (mitotic) cell must make sure the DNA strands are appropriately repaired by ligases prior to the completion of DNA synthesis and entry into mitotic chromosomal division and cell separation.

The eukaryotic cell cycle (G1, S, G2, M) contains two decision or check points that are vital to correct cell division. The one between G1 and S "evaluates" the cellular environment for capacity to support cell growth and division. The one between at the end of S in G2 prior to M "evaluates" completion of DNA "proof reading, " it looks as completion of ligation of fragments from DNA synthesis, including breaks caused by helicases. [It is probably a good idea to review a genetics or cell physiology text to review the cell cycle and the process of DNA replication in both mitosis and meiosis. The existence of checkpoints is covered in articles by Weinberg and _________.]

Note that in meiosis there are the same DNA synthesis hurdles plus breaks caused by the process underlying genetic reassortment, crossing-over, or synapsis, which requires DNA breakage and repair before packaging of chromosomes within gametes.

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Session 9.

1. Introduction to Prometheus, enrollment in Online part of offering, review of threaded discussion and other features of the site.

2. Do we need to review some topics on the syllabus as we are ahead of the topical list for the course? We have touched on some of these topics but it may be well to review them before too many sessions go by.

3. The utility of the listed toxicology sites has been duly noted. Some that are particularly helpful seem to be the FDA site and the ATSDR site.

4. The paradox of toxic intake by the oral route: that lower dose concentrations (and larger volumes) can sometimes be more toxic than higher dose concentrations [same amount of toxicant in either case, just different volumes].

Note the volume sensitive sphincter separating the stomach and the small intestine which has a very large absorptive layer. If the volume of the dose is increased this may lead to a faster emptying of the stomach, a lesser time for the HCl in the stomach to potentially inactivate the drug or toxicant, and more rapid exposure of the intestinal lining to a toxicant containing solution. In this situation even a lower total dose of compound may be more effective than a small concentrated dose.

Is there a secondary paradox? What if dietary insoluble fiber is present in the stomach at the same time as the dose?

What is insoluble fiber?

It tends to be macromolecular and will include polymers that are nondigestible for the species in question, e.g. cellulose (in nonherbivores), hemicellulose, pectin, silicates, and some minerals.

An increase in insoluble fiber will increase the transit rate through the gut, i.e., it will decrease the time needed to transit the gut. This effect is often protective as it reduces time of exposure to a toxicant. The effect is also enhanced by the presence of clean insoluble fiber as opposed to previously contaminated fiber. This is because clean fiber can efficiently adsorb toxicants from solution and effectively sequester them from absorption by the intestinal lining.

But contaminated fiber can also release toxicants for absorption. So there is an equilibrium between toxicants free in solution and those particulates or colloids, including insoluble fiber, within the digestive tract. Insoluble fiber can act as a sink or source for toxicants depending on its prior exposure to toxicant.

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Session 13.

1. Access to Prometheus with respect to documents and images was discussed.

2. Project progress is to the point of beginning writing. It has been suggested that the best beginning may be a detailed outline including pertinent citations. The early writing is begun the better the project will turn out.

3. Where are we with respect to reading and the syllabus? Students have read most of the material assigned through chapter 20 of Casarett and Doull. Keep reading, our discussions will touch on much of the textual content though in other orders than the text is written.

Reference back to the last several sessions emphasized recognition of the importance of the pharmacokinetic models in evaluating movement of toxicants into and through target systems and tissues. Note that Classical Models are data based and often require assumptions as to the uniformity of distribution or elimination of a toxicant. They tend to be simpler than Physiological Models but are less demanding to construct and evaluate as: "the appropriate physiological (e.g., blood flow rate, tissue volume) and biochemical (e.g., rate of biotransformation in a particular tissue) parameters [required for the Physiological Models] are often unknown or inexact, hampering meaningful physiologically based pharmacokinetic modeling." (Klaassen & Watkins, Casarett & Doull's Toxicology, Companion Handbook, p 134.) This latter restriction means that toxicologists must often run controls in parallel with their toxicant evaluations which will provide information on the basic processes involved. This adds to the value of toxicology studies but tends to slow them down. Note the Classical Models cannot predict tissue concentrations while well constructed Physiological Models can. The Physiological Model estimates are based on known or hypothesized biological processes, they require more basic data to compile, the math used to describe them is difficult (if requires calculus or iterative computer estimation of parameters), and the parameters involved are sometimes ill-defined.

The compartments used for the Physiological Models involve compartment size, flows into and out of these compartments, thermodynamic movement and partitioning between the compartments, and a consideration of the transport processes involved (e.g., perfusion versus diffusion limitations on transport between compartments).

Physiological Models are also used in allometrically scaling results among species. Look for a moment at what is meant by allometric scaling.

Allo- refers to "other," and allometry refers to the variation in growth of various parts of the body. Evolutionarily, allometric scaling, refers to the idea that certain uniformities or commonalities occurring among many species can be identified by measurements of appropriately chosen parameters. These include body mass, body surface area, body mass index (M/H^2), and basal metabolic rate, among others. As a central assumption in the extrapolation of animal test data to other species, including humans, it is reasonable to question the validity of this assumption. Note that many allometrist limit their considerations to comparisons of adult forms and often (stridently) limit considerations of scaling to spacial dimensions only. This rather static picture provides an adequate description for a number of situations such as life expectancy versus body mass in that a straight or monotonically increasing line can be demonstrated to hold across many species. It becomes more problematic for a parameter such as BMR where the association with body mass is a line only when body mass is raised to the 3/4 power. Note that volume can occupy and define such a formulation but inclusion of a time variable would be more adequate. But which aspect of time? Every organism grows from one or a small number of cells into an adult form. During that growth it accumulates a life experience, both good and bad, that helps define its final shape, form, and functions. That developmental time dimension is embedded in the cellular life history including numbers of cell doublings and numbers of cell losses. It includes the impacts of early and late toxicant exposures and nutrient limitations.

If an organism is large like a human, it tends to live a long time, have a long gestational period, and functions at a limited metabolic rate. A small organism tends to live a short time, has a short gestational period, and functions at a high metabolic rate. The number of cell doublings involved in making the larger organism is correspondingly larger than the number required for the small organism. During the longer lifespan of the larger animal additional cell generations add to the total number of cells that comprise that organism over a lifetime. Such an excess of cellular divisions means that the larger organism must cope with more potential problems associated with mutation or chromosomal nondisjunction whether caused by endogenous or exogenous factors. Either the larger organism will have more and/or more efficient cellular repair processes and/or toxicant degradation capacity or it will experience higher rates of mutationally induced disease (e.g., cancers) relative to the smaller species. This may have a large impact on the suitability of testing data generated in small species for application to large species (and, in fact, is part of the reason there are large 10-100 fold safety factors normally incorporated into exposure or dose limitations for humans or non-target species).

Note, however, that the short lifetime of the smaller species also means that all its cells are being exposed to their environment for, on average, a much shorter time than are the cells of the larger species. Thus, the risks of exposure, on a per cell basis, may be lower in the smaller species. This would tend to mean that the individual cells of the larger species would be inherently less prone to insult than those of the smaller species. Larger body mass also tends to have a shielding effect on the core cells.

In short, reasonable doubt must be exercised when attempting to allometrically scale data from small test animals to animals, including humans, who are of regulatory concern. That such scaling is potentially useful is unquestioned, that it suffers from many potential problems can be seen by the simple discussion above.

4. Generalized Types of Toxicity (Are current models adequate?)

1. Carcinogenesis

2. Genetic

3. Developmental

4. Other?

Environmental/Habitat (e.g., degradation of the environment yields a secondary impact on the organisms in question.)

Dietary/Nutrient (e.g., secondary plant metabolites may, for example, generate problems of absorption of nutrients due to the presence of toxins)

Carcinogenesis models:

The definitions given by C&D tend to rule out endogenous hormones as being classed as carcinogens unless they are exogenously administered. But what about endogenous overproduction of trophic or growth factors? Or incidental exposures through food and the environment?

The toxicology text provides a disappointingly naive discussion of endocrine systems given the similarities of toxicology, pharmacology, and endocrinology with respect to methods and many of the central concepts concerning mechanisms of action, chemical kinetics, clearance, and general physiological function.

The current carcinogenesis model looks at three stages in the "natural history of neoplasia"

1. Initiation

2. Promotion

3. Progression

In which a chemical may play a role in the actual induction of neoplastic transformation in the first cell involved. It may play a role in preserving that cell and making certain it can continue to divide. Or it may play a role in allowing the initial cell mass to invade other tissue and/or to metastasize to other sites. If the model is correct there should be examples of chemicals among the thousands examined that can be identified with each of these steps. Yet there are few, if any, that can unequivocally be assigned to initiation and progression. This would suggest there may be a problem with the specification or design of the current model.