Stress and dose interactions; Diet as modulator or mode of exposure; Developmental status/age and toxicity; C 4, 10, 30, 34

Course Comments

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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, 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.

Note that stress enhances both VP output and constrictions of blood flow to the kidney juxtaglomerular 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.

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.: New York, NY, 235‑253.

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.

Oxidative stress and radiation:

http://www.sysbio.org/sysbio/stress/

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 New England.

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.