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OCR for page 46
4
Principles of Toxicology in
the Context of Aging
The science of toxicology has a dual nature. It is the study
of mechanisms by which environmental agents exert their toxic
effects, and it is the empirical definition of the magnitude of tox-
icity of such agents and the risk they present, to exposed human
populations.* Toxicology, more than most disciplines of biology,
depends on comparison of experimental results with large data
bases acquired through the application of standard test protocols
to large numbers of chemicals and types of radiation.
To study both the possible exacerbation of aging processes by
environmental agents and the potential of increased toxicity for el-
derly people, mechanisms of aging must be considered with respect
to their similarity to mechanisms of toxicity of known toxic agents.
In addition, tests that will permit the screening of environmental
agents for their potential to interact with aging processes or to
present an increased hazard to the aged will need to be identified.
Once such potentially interacting agents are identified, they must
be evaluated in whole-animal systems that are appropriate surro-
gates for human response. The species selected must not only be
*For detailed descriptions of general toxicology, see Hayes (1982) and
Klaassen et al. (1986~. More specifically, Williams et al. (1987) have dis-
cussed the toxicologic perspective of the relationships between aging and the
environment.
46
OCR for page 47
PRINCIPLES OF TOXICOLOGY IN AGING
47
appropriate with respect to aging, but must also be representative
of human response for any particular chern~cal tested. Identifi-
cation of common mechanisms and creation of test systems must
both take place in the context of the existing data bases.
Some people view aging as a toxic process, and indeed some
age-related functional changes mimic toxic processes. Aging is
clearly associated with alterations in homeostasis and in organ
and cellular integrity; many changes of aging resemble those in-
duced by toxicants. Thus, aging might well be associated with
alterations in metabolic states that lead to the body's formation
of toxic molecules or to alterations in the normal regulation of con-
centrations of various natural substances in the body. Toxicologic
research has found that even the most unlikely substances can be
implicated in tissue damage if their concentrations deviate enough
from normal.
Nutrition underscores the usefulness of considering aging as
a toxic end point. For example, both deficiency and excess of
vitamin B6 can lead to necrologic damage (and these changes
superficially resemble those accompanying aging).
Another reason that aging can be usefully explored as a toxic
process in its own right is that aging itself is probably the primary
cause of or a cofactor in all age-associated disease. Furthermore,
aging cannot be readily arrested and is present during any whole-
animal experiment. Thus, all toxic processes induced by external
agents can interact with aging processes, and the possible impact
of this interaction would be greater with chronic exposures or toxic
effects whose latency includes a substantial portion of the life span.
If aging is a toxic process, can it be mimicked by chemical or
physical agents? The full answer to the question occupies much of
this discussion, but the simple answer is that many agents present
in the environment can, at high doses, induce some pathologic
changes and physiologic deteriorations similar to those observed
in the aged. This ~ a limited form of mimicry, because a given
agent generally produces only a few aspects of aging in only one
tissue (or at most several). Only a few agents, such as ionizing
radiation, cause damage throughout the body.
The absence of universal mimicry of aging by toxic agents,
even in a given tissue, is an unportant observation that must not
be overinterpreted. A toxic agent might produce molecular lesions
similar to aging-induced lesions. But the experience of toxicology
is that the manifestation of molecular or cellular damage at the
OCR for page 48
48
AGING IN TODAY'S ENVIRON~NT
tissue level ~ not rigorously specific to the toxic agent. Diverse
agents that are believed to produce the same kmds of damage at
the cellular or molecular level sometimes induce different kinds
of damage at the tissue level. Conversely, diverse toxic agents
sometimes produce similar pathologic effects; that is, the processes
that manifest some effects can be triggered by different agents.
All tissues deteriorate during aging. The observation of the
breadth of the effects of aging requires a similarly broad toxicologic
approach. Special attention must be directed toward evaluating
data bases for long-term whole-animal bioassays, because these
toxicologic evaluations have been used to evaluate the results of
prolonged exposure to toxic agents at low concentrations, including
environmentally import ant agents, and they constitute a major
too} for quantitative risk assessment.
Neurotoxicology and immunotoxicology should also be em-
phasized. Most environmental agents have not been screened for
neurotoxic and immunotoxic effects, and some neurologic and im-
munologic deficits of unknown etiology are observed in a large
proportion of the aged.
From the toxicologic perspective, two general problems pre-
sent the greatest concern in the consideration of environmental
relationships with aging. First, the mechanisms of aging are un-
known, so toxicologic methods of detecting perturbations in the
essential biology of the aging processes are not readily feasible.
Second, the empirical approach is thwarted because there are no
comparative data to validate any test proposed to detect agents
that specifically affect aging or the aged.
One approach to the two problems is to discern the mecha-
nisms of aging and search for agents that specifically affect aging
or the aged. Much effort has been expended on the former; great
resources would be needed for the latter. One purpose of this
report is to propose the most econorn~cal and scientifically sound
plan for the overall approach to the two problems.
In addition to aspects of conventional toxicology that appear
most relevant to the relationship of chemical toxicity and aging,
three new types of questions must be addressed:
.
How can agents that might interact with the agmg pros
cesses to increase or hasten the appearance of pathologic effects
or reduced resilience in the aged be identified? Certainly, the stan-
.
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PRINCIPLES OF TOXICOLOGY IN AGING
49
card long-term bioassay could detect large age-associated patho-
logic changes, but it is not clear whether it can mimic exposures
that are inherent in the human environment and absent from the
pristine laboratory environment of the surrogate animal.
~ How can acute or subacute exposures in utero, during de-
velopment, or during maturity that sensitize the exposed to other
agents be identified? For example, in multistage carcinogenesis,
exposure to a cancer initiator can sensitize animals to later ex-
posure to a cancer promoter. The problem of whether similar
mechanisms induce any of the wide spectrum of pathologic ef-
fects observed in the aged is extremely difficult to approach with
standard toxicologic tests.
~ How can environmental agents, particularly pharmaceuti-
cals, that hold increased hazard for the aged be identified? Most
toxicity Is measurer] in young animals, so a more specific question
is whether testing performed in young anunals can predict the
response of old animals. If the responses of young and old animals
to a given agent are qualitatively similar, but quantitatively differ-
ent, the application of a safety factor knight permit the data from
young anneals to be modified for predicting risk in old animals. If
the mechanism of action differs, such extrapolation would not be
possible; testing with old animals, and the attendant expense and
absence of comparative data, would then be necessary.
The remainder of this chapter summarizes the general con-
cepts of toxicology (i.e., absorption, distribution, metabolism, and
elimination), the effects of chemicals in the body as a function of
age, mechanisms of toxicity at various levels of physiologic organi-
zation, and the importance of pharmacogenetics, biomarkers, and
toxicity testing.
CHEMICAL FATE AND EFFECT
Chemicals enter the body by inhalation, ingestion, and con-
tact with the skin. They can act at the local site of contact, or
they can be absorbed, enter the bloodstream, and be transported
to act at other sites. Toxic agents are eliminated from the blood
by biological transformation and by excretion or accumulation at
various sites. The liver is active in biotransforming toxic sum
stances; however, enzymes in the kidneys, lungs, gastrointestinal
tract, skin, and other tissues can also metabolize toxicants. Some
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50
AGING IN TODAY'S ENVIRONMENT
toxicants accumulate in organs or tissues, where they might or
might not produce toxic effects. Most toxicants are eliminated
in the urine via the kidney or in the bile via the liver, although
some volatile toxicants can be eliminated in the exhaler] air via
the lungs.
Most of the research on the effects of old age on the absorb
tion, distribution, metabolism, and elimination of chemicals has
focused on drugs, rather than on toxic environmental substances.
Many toxic chemicals are converted to less toxic, or in some cases
more toxic, chern~cals by the same pathways that are responsible
for the biotransformation of drugs, so data on drugs have rele-
vance for toxicologic concerns. For information on aging and drug
disposition, several comprehensive reviews of geriatric pharmacol-
ogy are available (Greenblatt et al., 1982; Schmucker, 1978, 1985;
Vestal and Dawson, 1985~.
In contrast with the data on pharmaceutical agents, which
are based on studies of both animals and humans, virtually all
the data on toxicants have been obtained from experimental ani-
mals. Furthermore, as documented in a monograph by Calabrese
(1986), the human data and many of the animal data are related to
neonatal and young subjects. Of particular interest is the possible
influence of age on the detoxification of carcinogens, on the con-
version of procarcinogens to carcinogens, and on the interaction of
carcinogens with potential inducing agents.
Age-related changes in carcinogen metabolism in young and
old animals have recently been reviewed by Birnbaum (1987), who
concluded that the available data were both conflicting and sparse.
The results of metabolic studies depend on substrate, species, gen-
der, and even strain. Age-related changes in the monoxygenase
components of nonhepatic tissues have hardly been examined.
Thus, although it has been tempting to hypothesize that age-
dependent changes in carcinogen metabolism might contribute to
the increased incidence of cancer with aging, this is an oversimpli-
fication. For specific compounds, biotransformation might differ
with age in a given tissue, but the differences might not result in a
greater concentration or longer duration of action of carcinogenic
chemicals or reactive metabolites at vulnerable target sites.
Although many, but not all, animal studies have suggested
that aging is not associated with a loss of the capacity of oxidative
metabolism to respond to inducers such as barbiturates, poly-
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PRINCIPLES OF TOXICOLOGY IN AGING
51
cyclic hydrocarbons, and steroids the question has received less
attention from clinical investigators. In addition to metabolism,
such toxicok~netic measures as absorption, distribution, and renal
and biliary excretion and toxicodynamic deterrn~nants of tissue
sensitivity can change with age. Thus, an increased carcinogen
sensitivity in older persons might result from complex interactions
of many processes that have not been investigated systematically
in either humans or anunals.
Awareness of the effects of age on the absorption, distribution,
metabolism, and elimination of drugs can provide insight into the
mechanism of altered response to chemicals in general. Studies of
age differences in pharmacodynamics (biochemical and physiologic
effects of drugs and their mechanisms of action) must take into
account possible age differences in pharmacokinetics (absorption,
distribution, metabolism, ant! elimination). For example, studies
generally show that the elderly are more sensitive to the depressant
effects of neuroactive drugs (such as diazepam) (Giles et al., 1978;
Reidenberg et al., 1978) and the analgesic effects of narcotics (such
as morphine) (BelIville et al., 1971; Kaiko, 1980~; in contrast, the
in viva sensitivity of the heart to isoprotereno! and its antagonist
propranolo} appears to decline with age (London et al., 1976; Van
Brummelen et al., 1981; Vestal et al., 1979~. There is also evidence
that cellular biochern~cal responses to some drugs are altered with
aging.
Absorption
Absorption ~ the major process by which toxicants are trans-
ported across body membranes. The main sites of absorption of
toxic agents are the skin, lungs, and gastrointestinal tract. Many
toxicants can be absorbed through the skin and enter the blood-
stream. Chemical or physical injury and other circumstances can
increase the skin's permeability. Toxicants that are absorbed by
the lung are in the form of gases or solid or liquid aerosols. AM
sorption can be rapid and complete because the lungs have a large
surface area and a blood supply that is close to inhaled air in
the alveolar. A variety of environmental toxicants enter the food
chain and are absorbed from the gastrointestinal tract. Many
factors alter the gastrointestinal absorption of toxicants, including
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52
AGING IN TODAY'S ENVIRON
gastrointestinal motility, the physical and chemical properties of
the toxicant, and gastrointestinal content.
A number of physiologic alterations associated with old age
might be expected to affect absorption from the gastrointestinal
tract. They include an increase in gastric pH, a reduction in
intestinal blood flow, a reduction in the number of absorbing cells,
a decrease in gastrointestinal motility, and a slowing of gastric
emptying. An increase in gastric pH Fight affect the ionization
and solubility of some substances, but there are few specific data
on the range of pH values that can be encountered in the elderly
population and the effects of increased pH on bioavailability. Older
people have reduced gastrointestinal motility and slower emptying,
which might be expected to decrease the rate of absorption.
Some studies have shown an increase in the time to peak
plasma concentration after oral drug administration. This has
only minor clinical importance because the extent of absorption
did not differ between young and old subjects. Although the data
are sparse, most studies on drug absorption in the elderly do not
demonstrate a marked effect of age on the rate and extent of
absorption.
Distribution
Once a toxicant enters the bloodstream, it is available for
distribution throughout the body. Only free toxicants those not
bound to plasma proteins are able to enter other sites. Such
binding is of particular concern to toxicologists and medical scien-
tists, because toxicants bound to those proteins can be displaced
by other chemical agents and, once released, go to target organs
and produce injury there.
The distribution of toxicants depends on their ability to cross
cell membranes and on their affinity for various body components.
Toxicants vary widely in these two characteristics. Some do not
readily cross cell membranes and therefore have restricted distri-
bution. Others bind to various sites in the body, such as fat, liver,
kidneys, or bone. The major toxic action of a toxicant might take
place where it binds, but often it does not. In fact, binding sites
often serve as storage depots whose existence helps to protect the
body from the toxic action.
A variety of age-related changes can alter the volume of dim
tribution of substances throughout the body. Body composition
OCR for page 53
PRINCIPLES OF TOXICOLOGY IN AGING
53
is one of the most unport ant. Total body water (both in absolute
terms and as a percentage of body weight) is reduced by 1~15%
between the ages of 20 and 80 years; lean body mass in propor-
tion to body weight is also diminished with age, and body fat is
increased. These changes can be predicted to cause higher blood
concentrations of substances that are distributed mainly in body
water or lean body mass. Alterations in body fat can result in the
accumulation and prolongation of action of highly lipid-soluble
substances.
In addition, a decrease in serum albumin in the aged means
that greater amounts of substances that bind to serum albumin,
such as the anticonvubant phenytoin, will be free to diffuse into
body tissue. In contrast, serum a~-acid glycoprotein Is increased
in the elderly, and that reduces plasm~protein binding of weak
bases, such as the antidepressant imiprmnine and the antiarrhyth-
m~c drug lidocaine. Thus, because free-drug concentration is an
important determinant of drug distribution and elimination, al-
tered plasma-protein binding might be one cause of altered phar-
macokinetics in the aged. Available data suggest, however, that
disease and immobility have greater effects on alburn~n concentra-
tion than age itself.
Metabolism and El~n;nation
Some chemical agents that enter the body can remain as intact
molecules, but many are biologically transformed by metabolic
processes. Metabolic processes might involve simple and reversible
chemical or physical interactions that primarily affect transport
throughout the body and across membranes. In other cases,
metabolic processes can substantially alter the chemical nature
of the toxicant and create a more toxic or less toxic agent. The
metabolic processes can facilitate elimination from the body.
It has been useful to consider the metabolic processes as being
of two types. The first includes processes of oxidation, reduction,
or hydrolysis that primarily alter or add functional (reactive) moi-
eties to the molecule in question. The second includes chemical
reactions of pre-existing or newly formed functional groups on the
molecule with various endogenous chemicals (such as amino acids,
sulfate, and glucuronic acid) to form conjugates, or new chemicals.
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54
AGING IN TODAY'S ENVIRONMENT
The biosynthesis of these products often alters lipid or water sol-
ubility and ionization characteristics in ways that promote their
secretion and excretion.
The major routes for elimination of chern~cal agents from the
body are from the kidneys to urine, from the liver to bile to feces,
and from the lungs to exhaled air. Minor routes include secre-
tions from the body such as sweat, tears, saliva, mucus, digestive
juices, and milk- and hair, nails, and desquamated epithelial tis-
sue. As mentioned above, such factors as age and disease state
that interfere with kidney function or biliary excretion in the liver
can affect the toxic potential of chemical in the belly.
The kidney's excretory mechanisms include filtration in the
giomeruli and secretion and reabsorption in the renal tubules.
Elimination via the kidneys is thus a function of blood flow to the
kidneys, molecular volume relative to pore size of the giomerular
filter, physicochem~cal characteristics of the molecule that affect
membrane transport, and enzymatic or other systems that might
activate or facilitate secretion and reabsorption. Chemicals that
bind to large molecules, such as plasma proteins, might not be
eliminated by filtration and might be retained in the body for long
periods.
The liver is especially important as a route of elimination of
chemicals that are ingested, because most of the blooc] from the
gastrointestinal tract goes through the liver on its way to the
general circulation. The liver is in a unique position to metabo-
lize a chemical through its enzymatic systems and to secrete the
metabolites into the bile. Bile empties into the intestines, where
the chemical can either be further altered en cl reabsorbed or be
eliminated in the feces. Injury to the liver often affects biliary
function and impairs this route of elimination.
Elimination of chemicals from the body can be studied by
pharmacokinetic measurements, which are often based on the re-
maining concentration of a chemical or its metabolites in the blood
in relation to time. Such information usually provides a good es-
timate of the amount of the chemical available for toxic action.
However, storage of the chemical in tissue depots or the toxicity of
unmeasured, activated, intermediate chemical forms is sometimes
more important.
Processes of metabolism and elimination can be altered in
the elderly, but the evidence of altered hepatic drug metabolism
in humans is indirect. Autopsy studies have demonstrated that
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PRINCIPLES OF TOXICOLOGY IN AGING
55
liver mass in proportion to body weight declines after middle age
and that liver blood flow decreases with increasing age. For some
drugs, mainly those that undergo conjugation in the liver, there
is no clear effect of age on metabolism. However, age appears to
have a variable influence on the rates of metabolism of drugs that
are oxidized in the liver; most of the wide interindividual variation
in drug metabolism is more likely due to a variety of genetic and
environmental factors. Although the available data indicate no
effect of age on the inhibition of drug metabolism (Divoll et al.,
1982; Vestal et al., 1987), the equally limited data on the sus-
ceptibility of the elderly to induction of hepatic drug metabolism
conflict, some studies showing a decrease in the extent of induction
(Salem et al., 1978; Twom-Barina et al., 1984) and others no age
differences (Crowley et al., 1986; Pearson and Roberts, 1984~. The
effect of age on the induction and inhibition of drug metabolism,
as well as other drug interactions, requires further investigation.
Cigarette smoke contains polycyclic hydrocarbons, which are
potent inducers of some isozymes of cytochrome P-450. Most
cross-sectional studies have indicated that cigarette smoking is as-
sociated with less induction of biotransformation in elderly than
in young people. Whether this is intrinsic to aging or is the result
of selective mortality could be established only by longitudinal
studies, which have not been done. Nevertheless, the possibility
of the greatly reduced capacity of some elderly patients to r~e-
tabolize and eliminate drugs should be taken into consideration
when prescribing drugs for the elderly. This can be done either by
slightly reducing the dosage of potent drugs with low therapeutic
indexes or by watching the patients very carefully, to ensure thera-
peutic efficacy of prescribed medications and to detect undesirable
drug-related side effects early.
Studies in senescent experimental animals have shown reduced
hepatic enzyme activity, with resulting reduced capacity to me-
tabolize ([rugs and reduced hepatic enzyme induction. Most of
the data have been acquired in rats and mice, and the appar-
ent age-related changes might not be universal; species, strain,
and sex differences have been important variables in rodent stud-
ies. Studies with liver tissue from nonhuman primates have not
shown a significant decline in the content of cytochrome P-450 or
in the specific activity of NADPH cytochrome c (P-450) reduc-
tase (Schmucker and Wang, 1987~. Similarly, in vitro studies with
human liver tissue (also limited) have shown no effect of age on
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56
AGING IN TODAY'S ENVIRONMENT
microsomal drug-metabolizing activity (Woodhouse et al., 1984~.
The results conflict with those obtained in rodents, and to some
extent they conflict with in viva studies In humans. (In viva stud-
ies in nonhuman primates have not been performed.J They do
emphasize, however, the difficulties in extrapolating observations
made in experimental animals to humans and the need for clinical
investigation to evaluate the metabolism of drugs and chemicals
in humans.
One important chemical that is widely used as a drug by
persons of aIrnost all ages is alcohol. Alcohol is distributed into
total body water. In both rodents and humans, its volume of
distribution decreases with age (Vestal et aI., 1977; Wiberg et
al., 1971~. That decrease results in higher blood concentrations
after equivalent doses in the elderly than in the young. Acute
alcohol exposure inhibits and chronic exposure induces oxidative
drug metabolism in the normal liver. Alcohol itself is oxidizer!
predominantly by alcohol dehydrogenase, a cytoplasmic hepatic
enzyme, and to a lesser extent by the hepatic microsomal enzyme
system. Although the elderly are more sensitive to the behavioral
and cognitive ejects of alcohol, studies have not demonstrated
age differences in alcohol metabolism in humans (Vestal et al.,
1977~. Age has been shown to influence alcohol metabolism in
rats (Wiberg et al., 1970~.
Dietary composition is an important environmental determi-
nant of drug metabolism and drug toxicity (Alvares et al., 1979;
Campbell and Hayes, 1974~. Most studies have been conducted
in experimental animals (Campbell and Hayes, 1974~. Studies in
healthy human volunteers have shown that a low-carbohydrate,
high-protein diet (Kappas et al., 1976) and charcoal-broiled beef
(Kappas et al., 1978) increase the metabolism of antipyrine and
theophyIline, ant] dietary Brussels sprouts and cabbage (Pantuck
et al., 1979J increase the metabolism of antipyrine and phenacetin.
Charcoal-broiled beef contains benzota~pyrene and other poly-
cyclic hydrocarbons (Lijinsky and Shubik, 1964), which stimulate
the metabolism of benzota~pyrene in rat liver and placenta (Harri-
son and West, 1971~. Brussels sprouts, cabbage, turnips, broccoli,
cauliflower, and spinach incluce benzota~pyrene hydroxylase in the
rat (Wattenberg, 1971~. Indo! compounds in cabbage and Brussels
sprouts stimulate the metabolism of phenacetin, hexobarbital, and
7-ethoxycoumarin by rat intestine (Loub et al., 1975; Pantuck et
al., 1976~.
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PRINCIPLES OF TOXICOLOGY IN AGING
61
cell is the essential unit of aging, with manifestations at the tissue,
organ, and organism levels being sequelae of cellular deterioration.
Although aging research has been directed toward determining the
molecular damage or change that underlies cellular aging, it has
been unsuccessful in establishing a molecular etiology. Thus, in a
search for toxic agents that mimic aging, comparisons at the level
of the cell and tissue are appropriate.
Effects at the Tissue [eve]
Toxic action in intact animals is most often characterized
at the tissue level because altered tissue structure or function ~s
commonly observed. Such specificity might be due to toxic stress
at the site of exposure (e.g., Jung, skin, or gastrointestinal tract),
at the site of metabolic action (e.g., liver, brain, or kidney), or in
susceptible target cells. It is important, however, in understanding
the toxic mechanism acting at the tissue level to determine its
etiology at the cellular and molecular levels.
To return to the example of ionizing radiation, the induced
failure of the lining of the gut to perform its barrier function Is
the direct result of failure of stem cells to proliferate, which causes
sloughing of the intestinal mucosa. The failure of the stem cells to
proliferate is believed to proceed from the action of x-ray-induced
free radicals in their chromatin through mastitic inhibition. Thus,
when considering toxic effects related mechanistically to aging,
the basic goal must be to identify the sequence of events from
molecular changes to their sequelae at the level of the cell, the
tissue, and the organism.
PHA1~IACOGENETICS
In the course of investigations of drug metabolism and drug
disposition in humans, striking individual differences in response
to drugs and in ability to metabolize and drspose of drugs have
been noted. Some differences are due to environmental factors,
some to age-dependent (developmental) factors, others to genetic
factors, and many to complex interactions among those factors.
The scientific study of genetic factors that account for individual
differences in drug metabolism and drug response is called phar-
macogenetics. Pharmacogenetics Is of toxicologic importance in
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62
AGING IN TODAY'S ENVIRONMENT
that it reveals how some people, because of their genetic constitu-
tions, suffer toxic effects on exposure to xeno~biotics at doses well
tolerated by other people.
Of particular interest is the relationship between genetic dif-
ferences in rates of metabolism among subjects and the synthesis
of potentially toxic biotransformation products of a parent drug or
other chemical. In the past, the hepatic drug-metabolizing enzyme
system has been regarded as a detoxification system, because it
converts lipicl-soluble compounds that could otherwise remain in
the body indefinitely to more polar metabolites that are readily
excreted in urine. More recently, however, it has been recognized
that this enzyme system can produce potentially toxic, highly
reactive metabolites that combine with tissue macromolecules, in-
cluding DNA, to produce necrosis, immunologic reactivity, and
mutations (Sipes and Gandolfi, 1986~.
Qualitative differences among subjects in pathways of drug
metabolism and quantitative differences in the activities of the
enzymes that catalyze those reactions and pathways could be in-
volved in the regulation and control of such tissue damage. Thus,
genetic differences among subjects can render some more and oth-
ers less sensitive to the toxicity of different reactive metabolites.
As genetic entities are investigated In detail, the effects of age
on their expression often become apparent. Expression of a phe-
notype, and hence genetically modified (increased or decreased)
susceptibility to chemical agents, might occur only when a person
bearing genes that predispose to a particular kind of toxicity is first
exposed to the offending environmental chemical, and that might
not happen until late in life. Drugs are prescribed more commonly
to old than to young people, so the incidence of pharmacogeneti-
cally related drug toxicity might be expected to increase with age.
In addition, some genetically determined conditions are expressed
only relatively late In life, such ~ Huntington's chorea and some
forms of neuromuscular disease and diabetes mellitus.
Many factors have been systematically investigated and iden-
tified as contributing to the large interindividual variations that
characterize disposition and response to xenobiotics in humans
(Figure 4-1~. The factors include sex, time of day or season
of drug administration, presence of disease, hormonal and nu-
tritional status, stress, exposure to activators or inhibitors of the
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PRINCIPLES OF TOXICOLOGY IN AGING
Barometric
Sunilght- Pressure Dlseas~
Exercise ~ ~ ~ Infection
Immunizatlon
Lactation \~ ~ ~ ail ~ \
/ ~ ~ ~ ~ ~ ~ f Occupational
Exposures
regila~cy ~~¢ ~ Dmg9
\ Clrcadlan
\ and
~ Seasonal
VarlatlonS
Smoking ~ CONSTITUTION ~ Dietary
Tobacco or _' \ `' - ~ Factors
MarlJuana Aim, ~ Cardlo-
AIcoho! Intake Am Aft ~ ~ ~ ~~< ~~ ~ Vascular
Starvatlon of 5/ ~ ~ ~ At,, ~
Fever C5 ~ 2. 5 Immunological
~ ~ ~ ~ Functlon
Stress_ 5 Hepatic Functlon
Albumin _ Renal
Concentratlon Functlon
I Age ~ GENETIC
| I CONSTITUTION
63
Functlon
/
GantrolNtestl nal
Functlon
/
FIGURE 4-1 Schematic depiction of established or suspected environmental
factors that can alter genetically controlled rates of drug elimination. Lines
from each environmental factor to a central circle are wavy, to suggest that
modification of genetically controlled rates can occur at different magnitudes.
Such environmental effects need not occur directly at the genetic level. In
the outer circle, a line joins environmental factors, to suggest that several are
associated and interdependent, rather than independent. Source: Reprinted
with permission from Vesell (1982a).
hepatic microsomal drug-metabolizing enzymes (including chronic
administration of any of several hundred drugs), the status of the
heart, liver, kidneys, and endocrine organs and age (Conney et
al., 1971; Gillette, 1971; Vesell, 1982b).
In the past 20 years, genetic factors that directly affect xeno-
biotic response and disposition in humans have been discovered
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64
AGING IN TODAY'S ENVIRONMENT
(La Du, 1972; Omenn and Gelboin, 1984; Omenn and Motuisky,
1978; Vesell, 1969, 1971, 1973, 1984; Weinshilboum, 1984~. Some
60 factors have been identified, and many more probably exist. In
some people under some conditions, genetic factors are the major
or even sole cause of such interindividual differences. For example,
when age, sex, diet, and exposure to environmental chemicals that
activate or inhibit the hepatic drug-metabolizing enzyme system
remain constant among human subjects, large interinctividual vari-
ations in response to and disposition of xenobiotics remain. Many
of these variations have a genetic basis.
About 20%0 of patients in teaching hospitals in the United
States are there for treatment of adverse drug reactions, and 5-
30%o of the patients in these hospitals have at least one such
reaction (Cluffet al., 1965~. Adverse reactions to drugs occur more
frequently and with greater severity in old than in young patients
(Hall, 1982~. The normal wide disparity In individual patients'
responses to drugs is only one cause of adverse reactions, including
drug toxicity, but it constitutes an important contribution to this
major medical problem.
Table 4-1 lists 10 of the best-known and most intensively in-
vestigated pharmacogenetic conditions. In almost every one, a
toxic response ensues owing to drug accumulation secondary to a
marked reduction ~ the enzymatic conversion of the parent drug
to pharmacologically inactive metabolites. The function of the
enzyme is aberrant because of a point mutation in the gene that
controls its synthesis. Like most other inborn errors of metabolism,
the conditions listed in Table 4-1 are generally transmitted as au-
tosomal recessive traits. Thus, affected subjects inherit a mutant
allele from each of two phenotypically normal parents.
Geographic differences exist in the gene frequencies of several
pharmacogenetic conditions. Age effects have also been identified
for the acetylase polymorphism, but additional studies of other
pharmacogenetic entities in Table 4-1 need to be performed with
respect to age.
The history of the discovery of an age effect on the gene free
quency of the acetylation polymorphism is particularly instructive,
because it illustrates how careful the search for such age effects
must be. In the original observations of Evans et al. (1960), age
was not recognized as an important factor. Later, study of a small
number of subjects seemed to confirm that observation (Farah et
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PRINCIPLES OF TOXICOLOGY IN AGING
TABLE 4-1 Monogenic Metabolic Pharmacogenetic Conditions and Putative
Aberrant Enzymes
65
Conditiona
Acatalasia
Slow inactivation of isoniazid
Suxamethonium sensitivity or
atypical pseudocholinesterase
Phenytoin toxicity due to
deficient parahydroxylation
(autosomal dominant)
Bishydroxycoumarin sensitivity
(mode of inheritance unknown)
Acetophenetidin-induced
methemoglobinemia
Polymorphic serum aryl esterase
activity
Deficient N-hydroxylation of
amobarbital
Polymorphic hydroxylation of
debrisoquine in man
Polymorphic hydroxylation of
mephenytoin
Aberra I
Normal Location
Catalase in erythrocytes
N-Acetyl transferase in liver
Pseudocholinesterase in plasma
Mixed-function oxidase in liver
th at parahydroxyl at es phenytoinb
Mixed-function oxidase in liver
that hydroxyl at es b
bishydroxycoumarin
Mixed-function oxidase in liver
t hat de -ethyl at es acetophenet idinb
Serum aryl esterase
(paraoxonase)
Mixed-function oxidase in liver
that N-hydroxylates amobarbitalb
Mixed-function oxidase in liver
that 4-hydroxylates debrisoquine
Mixed-function oxidase in liver
that hydroxylates S-mephenytoinb
aAutosomal recessive, unless otherwise noted. Reprinted with permission from
Vesell (1982a).
bProbable but not fully established site of genetic defect.
al., 1977). But in 1983, Evans re-evaluated his original data and
reported an effect of both age and sex on plasma isoniazid concen-
trations (Iselius and Evans, 1983~. In 1984, the proportion of slow
acetylators was found to be significantly higher in older people,
but no sex distributions were reported (Gachalyi et al., 19843. In
1985, the age effect on the acetylator phenotype was confirmed,
but stated to occur only in males (PauIsen and NiTsson, 1985~.
The increased frequency of the sIow-acetylator phenotype with
age is of special interest, because the phenotype is also associated
with a markedly increased susceptibility to the development of
bladder cancer on chronic industrial exposure to arylamines and
hydrazines (Cartwright et al., 1982~. Another association between
susceptibility to cancer and a pharmacogenetic phenotype has
been claimed: extensive metabolizers of debrisoquin were stated
OCR for page 66
66
AGING IN TODAY'S ENVIRONMENT
to be more common in patients with bronchogenic carcinoma than
in age- and sex-matched controls (Ayesh et al., 1984~.
One implication of those variations among normal people is
that a given dose of a drug administered by a given route can be
toxic in one subject, therapeutic in another, and without pharma-
cologic effect in a third. The existence of these differences presents
a formidable challenge to a physician who must individualize ther-
apy (especially for drugs with low therapeutic indexes).
The therapeutic ramifications of large interindividual varia-
tions in the disposition of many drugs make it necessary to iden-
tify the mechanism responsible. Such identification is beset with
difficulties rooted in the extreme genetic and environmental het-
erogeneity of human beings. In laboratory animals such as rats
and mice, however, heterogeneity can be controlled. Each variable
can be manipulated independently, the quantitative contribution
of each to interindividual variations in drug disposition can be
studied, and dose-response curves can be constructed. In the last
decade, such studies have revealed many factors that can affect
drug disposition. In humans, pharmacogenetic conditions can be
categorized into those that affect how the body acts on drugs
(pharmacokinetic conditions) and those that affect how drugs act
on the body (pharmacodynamic conditions) (Vesell, 1973~.
Because most of the monogenetic (simple, single-gene) condi-
tions mentioned above are rare and make only a few drugs toxic,
they probably contribute in only a minor way to the major medical
problem of adverse drug reactions. However, another development
in pharmacogenetics suggests that genetic differences that directly
affect xenobiotic disposition play a prominent role in commonly en-
countered forms of drug toxicity. Large interindividual variations
that existed among unrelated people in response to pheny~buta-
zone, bishydroxycoumarin, antipyrine, halothane, ethanol, pheny-
toin, nortriptyline, or salicylate were absent in pairs of monozy-
gotic twins, but present in most, but not all, pairs of dizygotic
twins (Vesell, 1973, in press). The magnitude of interindividual
variations in rates of drug elimination among unrelated people was
a factor of 30 for nortriptyline, 10 for bishydroxycoumarin, 6 for
pheny~butazone and antipyrine, 3 for halothane, and 2 for ethanol.
The existence and operation of many environmental factors-
each with a different capability of altering the basal, genetically
controlled rate of drug disposition make it difficult to attribute
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PRINCIPLES OF TOXICOLOGY IN AGING
67
portions of the total interindiviclual variation to specific environ-
mental factors. The task of partitioning the total variation in
drug elimination among large heterogeneous populations is fur-
ther complicated by the close association of such seemingly pure
environmental factors as smoking and diet with other environmen-
tal factors, as well as with genetic factors.
Many environmental, developmental, nutritional, or endocrine
factors can influence the rate at which a person eliminates a drug.
In Figure 4-1 such factors are connecter] because they are often
associated with each other in a given subject rather than being
independent. In fact, they often interact dynamically to change a
subject's characteristic basal rates of drug absorption, distribution,
metabolism, excretion, or receptor interaction. Accordingly, the
effects of each of these factors on drug response can be complex
and can change with time even in the same subject (Vesell, 1980,
1982a,b).
BIOLOGIC MARKERS
A biologic marker ~ a biochemical, cellular, structural, or
functional indicator of an event in a biologic system or sample.
Biologic markers in humans, animals, or other biota can serve as
measures of exposure to or injury by a xenobiotic by indicating
internal or circulating dose, stored body burden, dose at a target
tissue, or the early onset of a pathologic effect.
The concept of biologic markers grew out of cancer research
that sought to identify the role of exogenous agents or host fac-
tors as causes of human cancer. Perera and Weinstein (1982)
defined molecular cancer epidemiology as an approach that com-
bined analytic epidemiology and molecular techniques to identify
carcinogens in human tissues, cells, or fluids and to measure early
morphologic, biochemical, or functional responses to carcinogens.
Lower and Kanarek (1982) described molecular epidemiology as
the measurement of molecular characteristics related to neoplastic
disease.
Since those early papers, several symposia and workshops have
been conducted to examine the use of biologic markers in disease
prevention. The ultimate goal of marker research is to improve
the predictive relationship between exposure, dose, and response.
A more thorough understanding of the role of markers will help to
prevent disease by more precisely assessing the magnitude of risk,
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68
AGING IN TODAY'S ENYIRO~NT
identifying high-risk groups or individuals, and providing early
warning of disease.
The development of cellular and molecular markers extend-
ing from exposure to the development of disease should provide a
powerful too! for the environmental health sciences. Markers that
indicate the presence of internal or biologically effective doses or
of incipient disease can be useful in hazard identification, that is,
as the qualitative step that causally associates an environmental
agent with an adverse effect. Markers can also be used to de-
terrn~ne dose-response relationships, particularly at the low doses
relevant to most environmental chemicals. A major role of markers
is to clarify the extent of human exposure in populations, the ex-
tent of individual exposure, and the proportion of high responders
or outliers among the human population (Fowle, 1984~.
. The use of biologic markers has raised a number of impor-
tant ethical issues (Ashford, 1986~. Among these is the concern
that biologic screening could encourage a shift from environmental
monitoring to human monitoring In the workplace. There is con-
cern that detection of a susceptibility marker, for example, could
be used to exclude a person from employment, ant! that focusing
on detecting susceptible populations and excluding them from the
workplace could replace efforts to remove toxic chemicals from the
workplace.
Markers can be distinguished on a continuum of time as mark-
ers of susceptibility, exposure, circulating internal dose, biolog-
ically effective dose (or dose at receptor site), and potential or
actual health impairment.
Markers of susceptibility indicate individual or population
physiologic differences that affect response to environmental
agents, regardless of exposure. They include differences in re-
ceptors, in metabolism, in immunogIobulins, or in organ reserve
capacity or other variations that lead to altered response to envi-
ronmental agents, including sex, age, physiologic state, and even
diet. For example, the absence of the enzyme a-1-antitrypsin is a
marker of susceptibility to chronic obstructive pulmonary disease.
Markers of exposure are biologic events or conditions that re-
veal information about external exposure, internal absorbed dose,
or Lose at the receptor site or site of toxic action. Markers of ~n-
ternal dose—indicating the amount of a material that is absorbed
into the organism—include such pharmacokinetic characteristics
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PRINCIPLES OF TOXICOLO G Y IN A GINO
69
as blood flow, capillary permeability, transport mechanisms, num-
ber of receptor sites, metabolism of the material, and route of
administration. Additional factors include data on structure and
stability of the material, peak or cumulative circulating dose, and
hal£life (Gibaldi and Perrier, 1982~. Markers of biologically ef-
fective dose include such target-organ characteristics as rates of
metabolic activation and detoxification, pre-existing susceptibili-
ties, and reserve capacity.
Markers of potential health impairment include early biologic
responses, such as alterations in the functions of target or nontar-
get tissue shortly after exposure. Later in the course of response to
a toxicant or after accumulation of high doses, markers of health
impairment include altered function of the affected tissue that
conic] be considered a precTinical state of disease.
Exposure to environmental pollutants can lead to uptake of
a biologically effective dose and ult~rnately to reversible or irre-
versible injury. Different types of markers can be used differently
to determine exposure, dose, or health impairment. However,
markers of these types are not always distinct from each other.
Thus, markers of exposures and markers of effects are often diffi-
cult to differentiate.
Because the body responds to injury with only a limited num-
ber of biochemical or cellular changes, an effect marker might not
always be specific for an individual pollutant.
TOXICITY TESTING
Toxicity testing is undertaken for two general purposes: to
characterize a particular chemical or physical agent so as to de-
termme its general or specific toxic properties, and to screen a
large number of agents for their likelihood of producing particular
toxic effects. In both cases, practical and scientific considerations
influence decisions about the nature and extent of testing. Each
decision entails a scientific judgment that is based both on known
toxic mechanisms and on data from tests of many chemicals with
a series of defined protocols. Practical restraints on resources and
time limit the testing of any particular agent. The complete test-
ing of an agent in the wide range of available standard protocols
could cost tens of millions of dollars. Few agents are theoretically
or practically important enough to justify such expenditures.
Toxicity-testing protocols can be divided into screening or
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Representative terms from entire chapter:
drug metabolism
70
AGING IN TODAY'S ENVIRONMENT
auxiliary tests and whole-animal tests. Screening or auxiliary
tests are usually short-term tests designed to identify a potential
for inducing specific toxic effects. Whol~animal tests can be short
term or long term and are designed to confirm or deny potential
toxicity and to assay risk quantitatively.
Two characteristics of screening tests determine their utility.
The first is whether the biologic process assayed by a test is iden-
tical with the process involved in the toxic effect of concern. For
instance, is mutation as observed in the Salmonella typhimurium
test a necessary and sufficient process in some or all types of car-
cinogenesis for which the test is used as a screen? The answer to
this specific question about the Ames assay is probably no; other
processes not detectable by this test are also relevant to the induc-
tion of cancer. Regardless, the Ames assay is a useful test because
it has the second characteristic: predictive value.
Predictive value is the ability of a test to predict the outcome
of another test or human response. It is a probabilistic measure
of correlation between the outcome of the simple test and the
~truth" as defined through whole-animal testing. Predictive value
is independent of essential biology. If, however, a short-term test
has power both in mimicking essential biology and in predicting
whole-animal response, it approaches the ideal.
In designing screening tests from the toxicologic perspective
of detecting agents that accelerate aging, the same criteria apply:
.
Does the proposed test mimic the essential biology of ag-
·
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PRINCIPLES OF TOXICOLOGY IN A GING
71
species will change, depending on the specific agent that is to be
tested.
Laboratory tests must, for practical reasons, be performed
with relatively small numbers of animals. Tests for environmen-
tal agents are generally performed at dosages much higher than
those to which human populations are exposed. Data from such
tests must be extrapolated to predict human responses to dosages
experienced in the environment. This extrapolation requires mea-
surement (or at lent prediction or assumption) of the shape of the
dos~response curve. Toxic effects are often classified according to
whether there is a threshoic! dose (below which no toxic effects
will be observed), or whether at low dosages toxic effects will be
produced in only a small proportion of exposed animals. This im-
portant distinction must also be considered for toxic interactions
with aging.
The measurement of dose-response relationships for reduction
in life span is difficult. Dose-response curves for life-shortening
are complex and not amenable to simple extrapolation to low
doses. One test of the hypothesis that radiation exposure induces
premature aging would be the demonstration that the onset of all
diseases is advanced to the same extent and by a factor related to
the degree of the shortening of the life span (National Research
Council, 1980b).
Another extrapolation that must be considered is that from
the laboratory to the real environment. Laboratory tests have
most commonly been performed with protocols wherein a single
agent is tested for its toxic effect; however, the human environment
contains multiple agents that can interact. In addition, there is
little evidence that interaction occurs between agents at the low
concentrations often found in the environment, but it is important
to consider the possibilities of such interactions. Interactions of
multiple agents are now being considered for their toxic effects,
and the results might guide similar efforts to study age-related
effects.
