Mammalian Toxicology, Session 1

Organization, passwords, e-mail addresses, student/instructor introductions, overview of course, projects. Definition and derivation of toxicology and sister sciences endocrinology and pharmacology; definition of toxins and toxicants; Key features of toxicology and study of toxicants; Modes of exposure, elimination, bioavailability, partition; C 1-3, 5, 7

Organization:

The course is handled via a series of 14 internet sessions along with a final exam. Projects will be generated by participants that will be peer reviewed by members of the class and will be posted to the course Prometheus site. Discussion will be handled via "threaded" e-mails/discussions on the Prometheus site. The instructor will post a topic or questions pertinent to the course material and students will respond by entering responses to the instructor's posting and/or to other student's comments. There will be tasks and searches also suggested for student actions either within the Discussion area or in Assignments or Announcements. Given the size of the course enrollment, I may try to get several subgroups to work together on some of the case studies that will be examined. Additional readings may also be posted in the Session or Assignment areas. The final exam for the course will be developed during the term, but the Instructor's preference is to give that exam via the Internet site. More will be posted concerning that during the term.

Passwords, e-mail addresses, etc.:

The course ID number is 1687 and the password is "poison". You should let me know if you are having problems accessing the site or the key areas: Outline, Syllabus, Sessions, Discussion, Assignments, Announcements. In case you need to contact me outside the course page my e-mail is kenneth.campbell@umb.edu . There will be an e-mail list of students taking the course so you can communicate as needed, so if you have not yet provided me with an e-mail address please do so as soon as possible. General information for handling the Prometheus site is located at http://www.lms.umb.edu . Information on technical requirements is at http://www.umassonline.net/tech/ and at http://www.conted.umb.edu/dl/faq/tech.html . Generally, if you've been able to download the information on the site so far, you probably are in good shape for the term. If you are trying to upload materials to the Discussion or other parts of the site always remember to hit the "submit" button at the bottom or side of the section you are typing into. If you are trying to upload files you have to upload a copy to the site before linking them to particular areas of the course site. In addition, if you are trying to upload files containing graphic elements, you must zip all the graphic files together with the text files and upload the zipped file to the course site before adding it to a particular part of the site. (Much of this is covered in the technical coverage of the use of the course site.)

Student/instructor introductions:

I have also asked each student to provide a brief personal introduction including an indication of why they are taking this course. In my own case, I have been with the University of Massachusetts at Boston for the past 21 years in the Department of Biology. My major course responsibilities until I started offering Mammalian Toxicology were an upper level undergraduate course in Endocrinology (including a laboratory) and graduate courses in Biomedical Tracers and Cellular and Molecular Endocrinology. I have also taught Biotechnology. These are all outgrowths of my undergraduate training in chemistry, biology, and math, my doctoral training in biochemistry, and my postdoctoral training in reproductive endocrinology and biology. My PhD dissertation on the effects of o,p'-DDT on the development of the steroidogenic tissues of the male rat also exposed me to a great deal of toxicology and many of the concepts engendered in pharmacology. So, although most of my current research continues to focus on endocrinology and reproductive biology, I am harking back to my scientific roots in teaching this class. Personally, I'm originally from Minnesota where I did my undergraduate degree, spent 12 years in Ann Arbor, Michigan, doing doctoral and postdoctoral work, and now live in northern Rhode Island about 50 minutes from the UMB campus. I just spent 7 months on sabbatical at the University of Washington working with people in the Departments of Anthropology, Obstetrics & Gynecology, and Zoology -- a great environment both physically and intellectually. But even while looking at the mountains out on the West Coast I anticipated and looked forward to this class and our interactions this term. I hope they meet your expectations. Welcome to this course!

Overview of course and projects:

The coverage will include a good deal of basic material at the beginning of the course. However, because I am not a traditional toxicologist, you will find some of my questions dealing with some of the basic assumptions in classical toxicology. It turns out that endocrinology and pharmacology are very closely related to toxicology in many of their approaches, techniques, and concepts. It also turns out that many of the mechanisms and problems turning up in toxicological studies are similar, if not identical, to those seen or explored previously by endocrinologists (and neurobiologists). Indeed, one of the newer areas of toxicological exploration in the past decade has been environmental hormones. As a result of these overlaps, we will spend some time on basics in endocrinology, reproduction, and development toward the middle and latter part of the term. If possible, I will also work in some coverage of regulatory elements as well because of the large role they play in the work of many toxicologists via the Food and Drug Administration and the Environmental Protection Agency.

Because we are working on the Internet, interchanges must be compatible with that medium. So you will see a lot of text, many URL links to websites, some documents scanned or downloaded from journals, and images whenever available to illustrate points. ( I haven't gotten far enough to incorporate music or motion pictures - just give me some time!) Use of the Web also means that your interactions have to be compatible with the Internet. So please write as clearly as you can and illustrate your points with graphics when possible - indeed, you may also learn a bit of computereez in taking this course. Assignments may take the form of document or Web searches ("treasure" hunts) or requests to assess data and arrive at conclusions that might have application in risk assessment or regulatory contexts. Any exams will not simply be multiple choice or true/false. They will also contain ample opportunity for essay responses.

Projects will focus on case studies and will have to generate HTML (computer compatible) documents. They will be submitted about a month prior to the end of the course and be subjected to peer reviews prior to final revision and submission to the Instructor for posting in the course site. If the enrollment exceeds 15, projects would be assigned to groups of 3 people who would jointly generate them and clearly indicate in their work-up what the contributions of each of these individuals has been.

Definitions and relations to sister sciences endocrinology and pharmacology:

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 not 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.

You should note the discussion in C&D covering the two types of dose-response curves generated by examining multiple exposures of single individuals to varying doses of a target chemical versus those generated by examining the statistical responses of many individuals to single but differing doses of a chemical. Note that in the end the curves tend to resemble one another but that in the population exposure situation, much of the variation in response derives not from the chemical, but from the particular genetic, nutritional, and environmental context of the subgroups within the population examined.

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

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.

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.

Key features of toxicology and study of toxicants:

Toxicology is a highly applied science. It borrows information, approaches, and techniques from many other areas to address its applied problems. Toxicology deals with chemically generated deviations from normalcy and therefore often has to do the basic science needed to determine what constitutes "normal" structure and function in the course of defining deviations from it. Thus, progress in toxicological studies can be slow. In studying toxicity it also has to devise and use biological assays that can demonstrate the presence of toxicity. But toxicity may exist at a variety of degrees and levels. A toxin may kill, it may permanently compromise function of a particular organ, it may temporarily compromise organ function, it may permanently mar a tissue, it may cause a temporary disfiguration, or it may inconvenience an organism via an impact on environmental essentials or aesthetics. Toxicity often differs among tissues and organs within a single individual and from individual to individual. Toxicology must generate tests that demonstrate the problems at each of these degrees of toxic insult. And it must produce information pertaining to the ranges of insult that might be expected within a population. Moreover, it needs to use that data to predict what levels of exposure may result in similar insults in other individuals and/or organisms. So it is not only descriptive, but predictive.

Toxicology deals with naturally occurring toxins as well as synthesized toxicants. In dealing with deleterious effects, its means of testing overlap with those of pharmacology when the latter is exploring undesired side effects. Its tests resemble those in other areas of biology because the same tools that are applied to elucidate normal biology are immediately adaptable to demonstrating deviations from normal. Dose-response testing for pharmacology and endocrinology are identical to those for toxicology except that toxicological testing often explores doses that are higher than those found effective in the other sciences.

Modes of exposure, elimination, bioavailability, partition:

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? 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.

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?

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.

Note 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.

We will revisit many of the concepts and details of exposure, absorption, partition, and elimination later in the course.

These are sketchy notes. They are meant to augment material in the C&D text, not to replace it. So keep reading!