Mammalian
Toxicology, Session 10
Stress
and dose interactions; Diet as modulator or mode of exposure; Developmental
status/age and toxicity; C 4, 10, 30, 34
Course
Comments
While I will post a
Discussion topic this week, you should spend some time reading and evaluating
all the various penultimate project files that have been posted. Compile
a reply with your comments and suggestions for each of the projects; this
should be done over the next week so people can respond to and incorporate
those comments in their final project submissions.
Stress
and Dose Interactions
Stress
Axes
What is stress
physiologically?
* 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, s uppressing 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.
Note that stress enhances both VP output and constrictions
of blood flow to the kidney juxtaglomerula r
apparatus leading to over production of renin, angiotensins, and aldosterone.
Pharmaceutical preparations targeted at stress-related or sedentary
life-style-related high blood pressure often inhibit angiotensin
converting enzyme (ACE inhibitors) so as to suppress the salt and water
retention caused by over activity of this axis. Diarrhetics
counter the salt retention actions of the axis while beta blockers target vascular
smooth muscule and increase renal perfusion.
Agents that augment salt and/or water retention accentuate any blood pressure
elevations and may stimulate chronic high blood pressure and the cardiovascular
accidents that accompany that condition.
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 signaling pathways and
intracellular signal cascades, they have the potential to either block or
exacerbate 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.
Reactive
Oxygen
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.:
Specific elements of the
concept are covered in the links provided below as well as in C&D Chapter
3. 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 signaling 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.
A definition of oxidative
stress:
http://www.bb.iastate.edu/~jat/glutchp.html
Role of
heat shock proteins.
http://ehpnet1.niehs.nih.gov/docs/1998/Suppl-6/1319-1323wijeweera/abstract.html
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
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
Apoptosis Induction
Ultimately many of these
stressors tend to lead to cell death via apoptosis by means of activating
mitochondrial damage and triggering of the cytochrome
c dependent cell death pathways, or via cell surface receptor-mediated pathways
(e.g., FAS/FASL, TRAIL). Tissue damage may thus be silent with
respect to the reticuloendothelial system.
However, if sufficiently widespread it can still lead to a diminution of tissue
function. Loss of cardiac capacity, especially on reperfusion with
well-oxygenated blood, would be an example of this kind of damage. Note
that developmentally and during tissue growth and repair, growth factors like
EGF or NGF often trigger production of proteins like Bcl
which block the induction of apoptosis via the Bax
mediated pathways. This makes sense when the environment of affected
cells is being exposed to high concentrations of oxygen via perfusion by blood
passing through new vessels and capillaries. Or when
active tissues are generating oxidative products such as lipid metabolites that
would otherwise cause cellular compromise.
So the end-products of stress
are not only temporary compromises in tissue function mediated by temporary
changes in tissue production of hormones or by alterations in the capacity to
respond to hormones. They may also include actual structural and
functional changes caused by the local intoxication effects of endogenously
produced reactive oxygen species. Although these may be
temporary and acute changes. They may also become chronic and
permanent via induction of apoptotic cell death in sufficiently large numbers
of cells in sensitive tissues.
Diet
as a Modulator or Mode of Exposure
Water
& Food as Vehicles of Exposure
While we often think of food
and water as simply nutriative, they also may contain
components or contaminants that may have toxic properties. Since no
animals can survive without water and nutrients, we have no choice but to
consume these materials. Our only recourse to limit their potential for
contributing to overall toxic insults is to eliminate or limit those components
or contaminants we know to be associated with toxic properties. This
stands in contrast to the approaches needed to limit toxic exposures in the
workplace and living environment where many toxic sources can be totally
avoided without consequence, and to the use of pharmaceuticals which undergo
extensive testing prior to manufacture and sale. The limitation of food
and water associated intoxication is a focus for public health with respect to
humans and a focus for environmental and ecotoxicology
with respect to other organisms including plants and animals. These foci
have led to a series of laws governing food production and sales (Federal Food,
Drug, and Cosmetic Act and amendments: http://www.uvm.edu/nusc/nusc237/ffdcatc.html
(or see) http://www.fda.gov/opacom/laws/lawtoc.htm;
Federal Insecticide, Fungicide, and Rodenticide Act
and amendments: http://www.epa.gov/region5/defs/html/fifra.htm)
and protection of water sources (Federal Water Pollution Control Act and
amendments: http://laws.fws.gov/lawsdigest/fwatrpo.html,
http://www.usdoj.gov/crt/cor/byagency/epa1251.htm
; Safe Drinking Water Act and amendments: http://www.epa.gov/safewater/sdwa/sdwa.html
, http://www4.law.cornell.edu/uscode/42/300f.html)
as well as to the establishment of government agencies (Food and Drug
Administration, Environmental Protection Agency) that implement these
laws. Much of this material is covered in C&D chapters 30 & 34.
For regulatory purposes food
is considered a mixture including both constituents and additives.
Constituents are legally unregulated. Additives are of three types:
unintentional additives which are unregulated if incidental; unintentional
additives which are regulated if they occur as a byproduct of normal handling
of the foodstuff (e.g., plastic monomers from containers); or, they are
intentional additives that were added to modify the quality, form, or handling
properties of the food to which they were added, in which instance they will be
either regulated or termed "generally recognized as safe" (GRAS).
Note that none of these
classifications are initially based on toxic qualities. They are a
practical breakdown of food components. Constituents may be every bit as toxic
as any intentional additive. For example, peanut proteins or oils may be
highly allergenic yet they are intrinsic elements of any foods derived from
peanuts. Likewise, the goitrogens found in some
yam species are chemicals produced by the yams themselves and the toxins
inherent to certain cycads or fishes are avoided only by careful preparation of
the foodstuffs. Vitamin A poisoning can occur with over-consumption of
liver, especially when taken raw from predatory species like polar bear.
Yet all these instances are nonregulated.
By contrast leaching of
metals like tin or lead or plastic monomers such as phthalate esters from food
containers or food handling equipment is regulated and monitored by the
regulating bodies.
These food contaminants
include: pesticides, packaging constituents, metals, animal drugs, food toxins
(of plant, animal, or mycotic origin), and bacterial
contaminants (including pathogens that are virulently infectious & those
that are highly enterotoxic). Screening for some of
these materials is the basis for many of the activities of the field staff of
the Food and Drug Administration, e.g., the meat inspectors in meat
processing plants and the fish inspectors on the docks and in the fish markets
in
Intentionally added
constituents are subdivided essentially by historic accident. Those
already in foodstuffs at the time of adoption of the controlling legislation
were segregated into two classes: those that were regulated and those that were
GRAS. Materials became GRAS if they had been used prior to the
legislation and had been demonstrated to be safe by testing, or, more likely
for most traditional additives, by lack of negative experience during prior use
in the public food supply, i.e., safe according to human epidemiological
evidence from prior use experience. Thus, many additives were
"grandfathered" in as GRAS on relatively slim evidence.
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 (e.g., cornstarch or flour to make sweetened cereals
less sticky)
Many of the chemicals or
mixtures that fall in these categories are covered by the GRAS
designation. But there is now recognition that this may not always be an
appropriate designation.
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. Recognition of these facts in light of the
Delaney clause (see below) has led to evaluation of some of the formerly GRAS
dyes and re-categorization of such compounds as under regulatory control.
As in many other areas of toxicant regulation and legislation the focus in food
legislation is on the prevention of acute effects and on carcinogenesis
potential. In current practice, this 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 continue to be used without
additional testing. 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.
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.
Diet as a
Modulator
Not only do contaminants in
food modify the consumer, constituents in food may modify the consumer's
exposure to and metabolism of certain toxicants. As we have stated earlier in
the course, 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, salicylic acid, nitrates).
Water
Water purity deals with a
host of potential toxicants: mineral contaminants, organic contaminant
chemicals and biological secondary metabolites, biological breakdown and waste
products, and protein, nucleic acid, and carbohydrate substitutents
deposited in the water supply by natural and manmade causes. While there
are natural contaminants in many water supplies, e.g., ferrous metals,
sulfides, plant and aquatic animal waste products, there are also pollutants
arising from anthropomorphic endeavors (farming, manufacturing, municipal
wastes, human wastes). Control of some of these is done using
flocculation or precipitation methods in waste and water treatment plants,
others are minimized by filtration over finely divided solids such as sand or
soil, and some are neutralized or killed by treatment of the water with
broad-spectrum chemical toxins, e.g., ozone, chlorine gas. In all
water regulations and purification processes the idea is to keep as many
non-water components as possible out of the water supply to minimize any toxic
loads in the consumers. The popular individual water filters now being
used in homes utilize finely divided charcoal or sintered silica or ion
exchange resins to remove whatever contaminants remain following public water
treatment and the piping of water through ducts that may serve as sources for
pollutants that had been removed prior to introduction into the distribution
system. Such reprocessing and filtration sytems
become all the more important when faced with the fact that the human
population relies for existence on approximately 1% of the world's total water
resources!
Developmental Stage/Age and Toxicity
The ability of organisms to
cope with toxic loads is a function of physiological status. That status
is a function of gender and age of development. We have previously covered some
of the issues during fetal development that arise because different
physiological systems mature at differing rates. Moreover, some of these
tissues change functions during development. There is little wonder that
targets for toxic insult shift over developmental time as do the capacities to counter
or repair the damage of such insults.
Note the various stages at which various organs and systems
develop and how these coincide with susceptibility to teratogens.
Physiological Change of Status in
Pregnancy
Now also consider the
physiological status during pregnancy. Not only has the normal endocrine
system shifted into overdrive with thyroid, adrenal, pancreatic, and hepatic
systems producing vastly more hormones, binding proteins and serum glucose than
in the non-pregnant state, but the fetoplacental unit
is also contributing significant amounts of unique hormones of its own, e.g.,
hCG, placental lactogen.
Moreover, the maternal body is being altered to handle the energetically
demanding processes of birth and lactation. Major shifts in energy
storage and metabolism are taking place during pregnancy and lactation.
The maternal body's capacity to deactivate or metabolize toxicants is also
being modified. Hepatic activities change relative to the nonpregnant state. Kidney blood flow and overall
clearance increases. The placenta both binds and metabolizes some
constituents on its own to say nothing of the fetus' own capacities for
toxicant binding and metabolism. Thus, it is not surprising to realize
that the pregnant female is not equivalent to a somewhat overweight,
non-pregnant female.
Physiological
Changes in Old Age
Similar considerations also
accompany individuals as they age. In these cases there are usually declines in
capacities to accommodate and repair toxicant insults at the cellular level as
well as systemic decreases in homeostatic system tone. Thus, it becomes
more difficult for the liver to detoxify lipophilic
toxicants or for the kidney to clear enough urine to remove toxicant
metabolites. Pancreatic and thyroid functions decline as does lung
function. Epidermal barriers become thinner and less well hydrated making
penetration of toxicants by the dermal route faster than previously.
Cellular cycle controls and immune surveillance for neoplastic
cells becomes less robust so apoptosis becomes unable to eliminate all possible
neoplastically transformed cells. Pathogen surveillance declines and immune protection against
allergens and pathogens decreases. Interestingly, some of the best
theories on what drives the aging process center on accumulations of unrepaired toxic insults (especially as initiated and
propagated via the reactive oxygen and reactive nitrogen production and
inactivation pathways) as the primary culprits. Age-related
mobilizations of fat reserves which frees previously non-bioavailable toxicant molecules also complicates the
overall picture of physiological status in the elderly. It is not at all
surprising that physicians often have a difficult time getting perscription dosages correct in older patients. Nor
is it surprising when older individuals succumb to toxicant loads that are
normally well-tolerated by the young or middle-aged adult population. And
while such considerations may be less easily observed among most other mammals
who often do not survive beyond the onset of old age,
the same processes seem to occur.
Overall, age, developmental,
and reproductive status do make very large contributions to physiological
capacity to absorb, distribute, metabolize, and excrete any toxicants to which
an organism is exposed. They greatly increase the variability of
sensitivities to dose seen within a population. And they make the
processes of administration or regulation much more challenging than they would
be in the absence of such intrapopulation
variability.
Discussion
Questions
Food Safety (QS2Q1)
11. What steps
are required for drug product clearance by the FDA? How are food
supplements handled by the same agency? Are there additional or
alternative rules for genetically engineered foods or drugs produced by
genetically engineered organisms? Do such rules seem adequate to assure
safety of humans using genetically engineered organisms or products? What
about environmental safety of these organisms or products?
Temporal Changes in Toxicity (QS2Q8)
18. Given an
increased knowledge of the metabolic pathways utilized by mammals to process
toxicants, how would you now design an experiment in which two neuroactive drugs were to be used repeatedly to test
particular brain circuits? Would any of this make any difference to your
interpretation of results in which there was an apparent decline with time in
the responsiveness to one of the drugs? Does this knowledge make you view
the use of multiple drugs in elderly patients any differently?
©
Kenneth L. Campbell, 2005