Mammalian Toxicology, Session 6

Projects (Scaling, Allometry, and Comparative Gut Physiology)

Projects

I strongly urge all of you to look over all the project summaries emailed or posted to the Prometheus site this past week.  Check out the ideas other groups are exploring so you can adapt them or consider them in your own explorations.

To fit everything in during the term, I'm going to take this opportunity to address three items that are often missed or forgotten in going through the other material in this course.  Scales or scaling has a direct bearing on both physical measurement of toxicants and on perceptions of what levels of intoxication or contamination are.  Allometry addresses some related issues with respect to extension of test results in one species to anticipations of what may be happening in another species. It also lays a foundation for looking at variability of toxicant impacts on diverse species.  Finally, a bit of information on Comparative Gut Physiology provides the basis for better understandings of why species vary so much with respect to sensitivity to various toxicants and toxins.

Scaling

The appended file is a summary of an article from a study by the US Geological Survey published in the March 15, 2002 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?  America's waterways contaminated by medications, personal care products

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?

Hormones are present in serum at levels ranging from ng/L to fg/L.  ATP is found in cell cytoplasm at mMolar concentrations. Albumin (MW about 66,000) circulates at 40 mg/mL.  Diabetics get worried if their blood glucose levels exceed 250 mg % (mg/dL) and they inject themselves with insulin measured in uU/mL.

My point is that we live in a scientific culture that has adopted Standard International units as a common currency, but we often fail to use that common currency.  It may be no more valid to use picomolar than part per trillion, but it is very difficult to readily envision either unit in comparison to the other even for the same compound.  Therefore, a useful exercise is to take one of these units for several compounds of differing molecular mass and see how the units scale relative to one another.  A g of water = 1 mL = 1cubic centimeter (and at standard temperature, 298K, and pressure, 1 atmosphere, provides the key conversion factor linking linear measurement, volume, and mass).

A mole of NaCl equals 58.5 grams and occupies about 44 mL of space, so1 millimole equals 0.0585 grams = 58.5 mg and occupies 0.044 mL or 44 uL of space (a little less than one large drop, or about two small drops of space).  A micromole of salt would weigh in at 58.5 ug and would occupy 0.044 uL = 44 nL (nearing the limit of measurement by available hand-held micropipetters or syringes). A drop to 1 nanomole would equal 58.5 ng (exceeding the limits of even good microbalances to differentiate from zero) and would occupy 44 pL (= 44 x 10^-12 L = 44 x 10^-9 cm³), a volume equivalent to a cube 3.5 x 10^-3cm (= 35 x 10^-3 mm = 35 um) on a side, the dimensions of a typical cell.  A picomole of salt would be 58.5 pg (about 10 times the mass of DNA in a single cell and nearly the mass of protein in a normal cell) and would occupy 44 fL (= 44 x 10^-15 L = 44 x 10^-3 pL = 44 x 10^-12 cm³), a cube now measuring 3.5 x 10^-4 cm (= 3.5 um) on a side, about the dimensions of a typical nucleus.  A femtomole of salt would be 58.5 fg (about the mass of the DNA in a typical human chromosome) and would occupy 44 attoL (= 44 x 10^-18 L = 44 x 10^-15 cm³), a cube that now is 3.5 x 10^-5 cm (= 0.35 um = 350 nm) on a side, now the order of size of a wavelength of blue light, the length of a collagen molecule (Koolman & Röhm, Color Atlas of Biochemistry, Thieme:New York, 1996, 63) or 10 to 30 times the volume of a typical globular protein.

Now if we take a mole of salt and place it into a liter of water to make a 1 M solution that solution weighs about 1000 g, so the 58.5 grams of salt comprise 5.85 % of the mass of the solution, or 5.85 parts per thousand.  A 1 mM solution, made by taking 1 millimole of salt and dissolving it in a liter of water has 58.5 mg of salt 1,000,000 mg of solution, 0.00585%, or 5.85 parts per million.  A 1 uM solution of salt would be 58.5 ug per 1,000,000,000 ug of solution, or 5.85 parts per billion.  And a 1 nM solution of salt would be 58.5 ng per liter equal to 5.85 parts per trillion.

Note that the hormone values I quoted were from ng/L to fg/L, so typical values there would be nM to attoM, or roughly ppt to parts per quintillion.  ATP would be found in ppm, and proteins would range as high as high uM levels.  Toxins and pollutants, then, when quoted as ppm, ppb, or ppt, are actually nearer the higher end of physiologically encountered molecular concentrations in comparison to normal eukaryotic organismal constituents.

So how good are our physical measurement methods?  Well, colorimetric analysis or visual color reactions work to levels of uM (ppm), while light spectroscopy is often good to nM concentrations (ppb to ppt) and fluorescence spectroscopy works to pM or fM concentrations (ppt to parts per quintillion).  HPLC or gas chromatography often detect ng to pg quantitities which often translate to uM to pM concentrations (ppb to ppt).  Mass spectroscopy often works to attoM or even lower while some forms of fluorescence detection or electron microprobe analysis are capable of detecting single molecules of a given compound.  Immunoassays and radiometric analysis reach nM levels minimally (ppb) and fM concentrations under optimal conditions (pp quintillion).  Note that as methods have improved over the years we have gone from the point of being limited by physical measurements in our ability to determine the presence of toxicants, contaminants, or pollutants to the current situation where natural background begins to interfere with our abilities to set reasonable exposure limits that reflect real toxic risk.

You may find the following links useful in translating various values:

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

A set of converters, calculators, and estimators: http://www.wolinskyweb.com/measure.htm

And one of the most comprehensive resources for conversions available: http://www-sci.lib.uci.edu/HSG/Ref.html

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/H²), and basal metabolic rate (BMR), 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 allometrists 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 for insults such as radiation or oxidative compounds.  On the other hand, small species have a much larger surface area to mass ratio making them potentially more susceptible to externally applied agents.

Taken together these considerations should help in interpreting the variations observed among mammalian species which range in size from the shrew at a few grams to the blue whale at more than 100,000 kg.  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.

Comparative Gut Physiology

But allometric scaling is only a part of the complexities in interpreting toxicological information.  A review of comparative gut physiology across mammalian species will also prove helpful.  Illustrations of various gut anatomy arrangements or discussions of the various forms of gut physiology can be found at:

http://www.newveg.av.org/anatomy.htm (a primer for comparative anatomy)

http://helios.bto.ed.ac.uk/bto/biology/compneut/Notes/Supply/Digestion/DigestionPlantMat/MicrobialFerm/fermentation.htm (ruminant digestive physiology)

http://cnrit.tamu.edu/rlem/textbook/Chapter2.htm (digestion in range animals)

http://helios.bto.ed.ac.uk/bto/biology/compneut/Notes/Supply/Digestion/Introduction/intro.htm

(introduction to comparative gut physiology)

http://arbl.cvmbs.colostate.edu/hbooks/pathphys/digestion/herbivores/ (digestive physiology of herbivores)

http://trc.ucdavis.edu/mjguinan/apc100/modules/ (comparative anatomy and physiology)

while a thorough discussion of comparative anatomy and physiology are to be found in Stevens and Hume, Comparative Physiology of the Vertebrate Digestive System, 2nd Ed, Cambridge University Press: Cambridge, UK, 1995.

Humans resemble omnivores like pig and rat more than they do ruminants like sheep or hindgut fermenters like rabbits or horses. 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 systems 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., a recent best-seller Ghosts of the Past described the evolutionary interactions between plants with large fruits and the Pleistocene megafauna such as ground sloths, camelids, and horses).  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 intestinal tract.  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.  (A good link 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 can be found at:

http://www.orst.edu/instruct/ans311/311cl05.htm

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 ingestion but deprive them of the calories normally released from the cellulose in the hindgut thereby leading to calory 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.  This contrasts with the mouse or most other commonly used test species.

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.

Maturation of the gut plays a major role in determining just how problematic some toxicants 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).

While C&D focus on the human as the usual species of toxicological concern, they also note some of the peculiarities noted above for test species.  They do not extrapolate the magnitude of these problems to other domestic and wild species that may also be of concern.  They do however note in Chapter 6 (p 145) how the pathways for metabolism and clearance of chemicals introduced into the body are not only functions of the enzymes present in particular tissues, they are also a function of the particular size and quality of the tissues present.  These differ among species and as one result they mean that it is virtually impossible to replicate the pharmacokinetics or toxicokinetics of a chemical in an in vitro model; an intact animal is still the best test system for evaluation of toxins or toxicants.

Gut Physiology (QS3Q2)

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

Macromolecular Chemistry (QS3Q3)

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