Influence of Body Weight, Diet, and Stress on Aging, Survival and
Pathological Endpoints in Rodents: Implications for Toxicity Testing and Risk
Assessment
Julian E. A. Leakey1*, John E. Seng 2, and William T.
Allaben 1
1 FDA’s National Center for
Toxicological Research (NCTR), Jefferson, Arkansas 72079
* Corresponding Author’s email:
julian.leakey@fda.hhs.gov
2 Discovery and Development Services, Charles River Laboratories –
Redfield, 100 E. Boone Street, Redfield, Arkansas 72132
Abstract:
Dietary restriction in rodents has been repeatedly shown to increase lifespan
while reducing the severity and retarding the onset of both spontaneous and
chemically induced neoplasms. These effects of dietary restriction are
associated with a spectrum of biochemical and physiological changes that
characterize the organism's adaptation to reduced caloric intake and provide the
mechanistic basis for dietary restriction's effect on longevity. Evidence
suggests that the primary adaptation appears to be a rhythmic hypercorticism in
the absence of elevated ACTH levels. This characteristic hypercorticism evokes a
spectrum of responses including: decreased glucose uptake and metabolism by
peripheral tissues, decreased mitogenic response coupled with increased rates of
apoptosis, reduced inflammatory response, reduced oxidative damage to proteins
and DNA, reduced reproductive capacity, and altered drug metabolizing enzyme
expression. The net effect of these changes is to: (1) decrease growth and
metabolism in peripheral tissues to spare energy for central functions and (2)
increase the organism's capacity to withstand stress and chemical toxicity.
These adaptations suggest an evolutionary mechanism that provides rodents with
an adaptive advantage in conditions of fluctuating food supply. During periods
of abundance, body growth and fecundity are favored over endurance and
longevity. Conversely, during periods of famine, reproductive performance and
growth are sacrificed to ensure survival of individuals to breed in better
times. This phenomenon has been observed in rodent populations that are used in
toxicity testing. Improvements in animal husbandry and nutrition, coupled with
selective breeding for growth and fecundity, resulted in several strains
exhibiting larger animals with reduced survival and increased incidence of
background lesions. Mechanistic data from dietary restriction studies suggest
that these large animals will also be more susceptible to chemically induced
toxicity, thus creating problems in comparing tests performed on animals of
different weights and in comparing data generated today with the historical
database. The rational use of dietary restriction to control body weight to
within preset guidelines was proposed as a possible way of alleviating this
problem. Recent data from studies testing this paradigm have demonstrated that
dietary control not only can increase animal survival in two-year studies but
also can increase bioassay sensitivity.
Introduction
Dietary restriction has been repeatedly shown in rodents to increase
maximally achievable life span and to decrease the incidence and proliferative
rate of spontaneous and chemically induced neoplasia (1,2). During the last
decade significant new information became available on the biochemical and
molecular mechanisms through which dietary restriction influences cancer rates
and aging (3-8). Nutrient stress, which is characterized by elevated
glucocorticoid levels in the absence of elevated ACTH or inflammatory cytokines,
appears to play a central role in mediating the effects of dietary restriction
(3,9-11) and has certain similarities to the stress response elicited by many
chemicals when administered to rodents at their maximally tolerated dose (3).
While it has been known for over 60 years that survival and neoplasm
incidence in laboratory rodents is influenced profoundly by caloric intake and
body weight (12), it was only during the last decade that diet and body weight
have become major issues in the design and interpretation of animal toxicity and
carcinogenicity studies. Attention to these issues was precipitated by the
observation, during the early 1990s, that mean life span of rodents, which were
commonly used in cancer bioassays had been steadily decreasing, concurrently
with an increase in mean body weight and in the background incidence of
neoplastic and other degenerative diseases. For several strains it had reached a
point where assay interpretation was being compromised due to insufficient
animals surviving to the end of the study (13-15). Various symposia addressed
this issue and generally recommended that some form of dietary control, either
through dietary restriction or new diet formulations, be used to maintain
animals within a healthy weight range during toxicity testing (16,16-20).
Several approaches to dietary control have now been tested, and there has been a
trend among rodent breeding companies to select for smaller animals. This paper
reviews and updates the current state of knowledge on how dietary restriction
evokes its beneficial effects on aging and disease and describes the relative
success of dietary control techniques in increasing survival while decreasing
variability and background neoplasia rates in laboratory rodents.
Hypercorticism - An Adaptive Response to Nutrient Stress
Although the precise mechanisms by which dietary restriction evokes its
beneficial effects on disease and longevity have not been fully determined, it
is becoming evident that glucocorticoid hormones play a significant role in
mediating these effects (10,21). It was first reported over fifty years ago that
caloric restriction resulted in adrenal hypertrophy (22), and during the last
few years a number of laboratories have demonstrated increased corticosterone
concentrations in serum from dietary restricted rats and mice (10,21,23-30).
The mechanism controlling the adaptive response to reduced caloric intake
involves the complex, dynamic interplay between the hormones that control energy
balance, appetite, cell proliferation and apoptosis, stress response, metabolic
rate, inflammation, and repair systems (3,21) [See Figure 1 following this
paragraph]. Glucocorticoids and insulin appear to play a reciprocal role as the
major mediators of energy balance and glucose homeostasis in mammals (31-33).
Serum corticosterone levels rise in response to hypoglycemia and increase blood
glucose levels by inhibiting glucose transport into peripheral tissues while
increasing gluconeogenesis and glucose output by the liver. In the hypoglycemic
state, corticosterone also stimulates appetite by inducing neuropeptide Y
production in the arcuate nucleus of the hypothalamus (34) and stimulates
lipolysis in adipose tissue, while reducing energy expenditure in other
peripheral tissues by decreasing thermogenesis and inhibiting the effects of
mitogenic and excitatory hormones (21,32). Conversely, in the hyperglycemic
state insulin levels rise and decrease blood glucose levels by stimulating
glucose uptake and glycogen synthesis in liver and muscle and by increasing
glucose uptake and lipogenesis in adipose tissue (31,32). Insulin also
stimulates leptin production in adipose tissue, which in turn decreases appetite
and increases metabolism and energy expenditure in peripheral tissues (35-38).
Glucocorticoid treatment also stimulates leptin production; but this is possibly
an indirect effect resulting from increased insulin levels, increased insulin
sensitivity or functional maturity of adipocytes (39). The leptin gene promoter
region does not contain glucocorticoid response elements; and during fasting
conditions, where glucocorticoid levels increase, while those of insulin
decrease plasma leptin levels also decrease (39,40). Thus, under normal
physiological conditions, a balanced opposing relationship exists between
insulin and corticosterone, which maintains blood glucose levels within the
normal physiological range (31).
Figure 1. Hormonal Control of Glucose and Energy Homeostasis
Energy homeostasis and physiological blood glucose levels are maintained
predominantly by the reciprocal actions of glucocorticoids and insulin. These
hormones in turn regulate mitogenesis and growth rate via growth hormone, IGF1
and DHEA. See text and (31) for references. Blue lines = response to caloric
deficit, red lines = response to caloric excess, black lines = classic stress
response. AVP = arginine vasopressin, SST = somatostatin, NPY = neuropeptide Y,
CRF = corticotropin releasing factor, CCK = cholecystokinin, DHEA =
dehydroepiandrosterone, and IGF1 = insulin-like growth factor 1.
Nutrient stress, such as fasting, starvation or insulin-induced hypoglycemia
results in elevated glucocorticoid levels, but unlike classic stress,
hypothalamic release of corticotropin-releasing factor (CRF) does not appear to
play a major role in initiating the glucocorticoid response (41-43). Rather
arginine vasopressin (AVP) plays the major role in the hypothalamus, and the
adrenal response to ACTH appears to be amplified by pancreatic polypeptide,
which is secreted by the pancreas during periods of hypoglycemic stress (44-46).
In addition, adrenal corticosterone secretion may be further increased by neural
stimulation via the adrenal medulla (47,48). This results in elevated
corticosterone concentrations in the absence of elevated ACTH (and, by
inference, CRF) in both starved (41) and calorically restricted (10,43) rats.
Under normal physiological conditions, once the hypoglycemic crisis has been
rectified, insulin levels will rise and, as suggested by in vitro
experiments (49), may down-regulate adrenal corticosterone secretion in favor of
dehydroepiandrosterone (DHEA) secretion. DHEA, like insulin is generally
anabolic in function, and it is reported to antagonize many of the effects of
glucocorticoids (50-53).
During pathological conditions such as Cushing's syndrome or prolonged,
excessive glucocorticoid therapy, natural feedback regulation is bypassed, and a
pathological hyperglycemia develops, which is characterized by concurrent
elevated insulin and glucocorticoid levels. Such conditions of hypercorticism
concurrent with hyperinsulinemia, if prolonged, would be expected to result in
pathological conditions such as atherosclerosis and mature-onset diabetes (54).
Classic stress appears to be primarily controlled by the
hypothalamic-pituitary-adrenal axis (HPA). CRF and AVP secretion from the
hypothalamus increase in response to interleukins or neuropeptides and stimulate
ACTH secretion by the pituitary (55). Thus, plasma concentrations of both CRF
and ACTH are increased in addition to serum corticosterone levels. CRF decreases
hyperphagia (56) and is pyrogenic and an inflammatory mediator (57).
Anti-Neoplastic Effects of Glucocorticoids
The net effects of hypercorticism resulting from nutrient stress are,
therefore, a reduction of glucose uptake and energy metabolism in peripheral
tissues. This, in itself, may provide a beneficial effect on aging and
carcinogenesis by reducing rates of intracellular glycoxidation and oxidative
damage from respiratory chain enzymes (58,59). However, the primary mechanism by
which glucocorticoids impact upon aging and degenerative disease may be through
their anti-mitotic and anti-inflammatory functions.
Anti-mitotic effects: Growth hormone and glucocorticoids are
mutually antagonistic in their effects on body growth (60) and wound healing
(61), and some of the anti-mitogenic effects of glucocorticoids are mediated
through changes in the hypothalamic-pituitary-liver growth hormone/IGF1 axis.
Glucocorticoids disrupt the pulsatile secretory profiles of growth hormone
(62,63) in rats and decrease hepatic IGF1 expression (64). They also antagonize
the proliferative effects of EGF and PDGF in various cell culture systems
(65,66) and antagonize the stimulatory effects of Luteinizing Hormone (LH) on
the adenyl cyclase/cAMP system in Leydig cells and possibly other endocrine
tissues (67,68). Although high glucocorticoid levels can cause atrophy of
skeletal muscle, they stimulate hypertrophy in cardiac muscle (69). This effect
is associated with alterations in expression of myosin isoforms resulting in the
high efficiency V3 isoform being favored over the low efficiency V1 isoform
(69,70). Thus, the anti-mitogenic effects of glucocorticoids appear to be
selective.
Apoptosis plays an important role in inhibiting tumor development by eliminating
damaged and genetically transformed cells from tumor susceptible tissues
(71-76). Apoptosis is characterized as differing from tissue necrosis in that
only selected cells are eliminated, and the resulting cell debris is immediately
phagocytized by adjacent cells so that an inflammatory response is not initiated
(77,78). Glucocorticoids induce apoptosis in lymphatic tissues (72), fibroblasts
(79) and, possibly, in mammary epithelium (77,80). Glucocorticoids may also
selectively mediate the effects of TGFß in stimulating apoptosis in
preneoplastic hepatocytes (58,66).
Anti-inflammatory effects: When used therapeutically, glucocorticoids
are extremely potent anti-inflammatory agents, which interact with practically
every stage of the inflammatory response (81). Although it was once proposed
that physiological levels of endogenous glucocorticoids stimulated the
inflammatory response, as part of the general adaptation to stress (82), it now
appears their physiological role during stress is to protect the organism from
an overstimulated inflammatory response (81,83). Glucocorticoids achieve this by
inhibiting the production of, or antagonizing the actions of, inflammatory
mediators such as prostaglandins, leukotrienes, interleukins, and atrial
natriuretic factor (3,21,81) [See Figure 2 following this paragraph]. Many of
the anti-inflammatory and anti-mitotic effects of glucocorticoids appear to be
mediated by the glucocorticoid-inducible protein, lipocortin 1 (81,84-87).
Lipocortin 1 (also known as annexin 1) is a glycosylated 37 kDa Ca2+-dependent
phospholipid binding protein, which inhibits phospholipase A2, a key
enzyme in the synthesis of inflammatory prostaglandins and leukotrienes from
arachidonic acid (81). In addition to directly inhibiting phospholipase A2
activity, lipocortin 1 recently has been shown to inhibit the EGF-mediated
phosphorylation of the cytosolic form of this enzyme (cPLA a)
(88,89). cPLAa is activated
by phosphorylation as part of a G-protein dependent, EGF-mediated mitogenic
response (88). Lipocortin 1 also mediates glucocorticoid feedback effects on the
HPA-axis by inhibiting both basal and interleukin-induced release of CRF and AVP
by the hypothalamus (55,84,90). Glucocorticoids also down-regulate mRNA
expression of several key inflammatory enzymes. These include 12-lipoxygenase
(91) and the inducible, but not constitutive, forms of prostaglandin synthase
(COX2) (92-95), nitric oxide synthase (iNOS) (95-97) and intestinal
phospholipase A2 (PLA2II) (98). These enzymes generally
are induced by endotoxins, tumor necrosis factor, interleukins, phorbol esters,
or growth factors. Although it is not known whether glucocorticoids directly or
indirectly repress transcription of these enzymes, Lipocortin 1 appears to
mediate glucocorticoid-mediated down-regulation of iNOS (99), but not COX2
(100).
Figure 2. Intracellular Actions of Glucocorticoids Mitogenic
Signal Transduction Pathways
Glucocorticoids downregulate NF- k
B and AP-1 signal transduction pathways at multiple levels. In many cell types
these pathways activate inflammatory or mitogenic responses and inhibit
apoptosis. In addition to transrepressing the interaction of activated NF-kB
and AP-1 complexes to their DNA response elements, glucocorticoids induce
proteins, which inhibit the activation of these complexes. Abbreviations used:
AA = arachidonic acid, AP-1 = activator protein 1 complex (the active complex is
composed of a dimer of a c-fos protein with a phosphorylated c-jun protein),
cPLAa = cytosolic
phospholipase A2a,
COX2 = prostaglandin synthetase (cyclooxygenase) 2, CXC = chemokines, EGF=epidermal
growth factor, GC = glucocorticoid, GR = glucocorticoid receptor, GRE =
glucocorticoid response element, IkB
= NFk B complex inhibitor
protein, Ik BK = IkB
kinase, IL-1 = interleukin 1, iNOS = inducible nitric oxide synthase, JNK = Jun
N-terminal kinase, LP-1 = lipocortin 1, LPS = lipopolysaccharide, LT =
leukotrienes, MPK-1 = mitogen activated protein kinase phosphatase 1
(inactivates JNK by dephosphorylation), NF-kB
= nuclear factor kappa B (the active complex is composed of a dimer of the P-50
and P65 proteins), PG = prostaglandins, Ps = proteasome complex which degrades
phosphorylated IkB, R =
plasma membrane receptors for cytokines and other inflammatory molecules, ROS =
reactive oxygen species, TNFa
= tumor necrosis factor alpha, TPA = 12-O-tetradecanoylphorbol-12-acetate, TRE =
TPA response element.
Lipocortin 1 has been proposed to be a mediator of glucocorticoid-induced
apoptosis. It is induced in apoptotic cells where it has been proposed to
inhibit recognition of the dying cells by macrophages (77). Lipocortin 1 is also
a substrate for transglutaminase. This enzyme is induced in apoptopic cells
where it catalyzes the covalent linkage of proteins. Covalently linked
lipocortin dimers can form polymers with other proteins during apoptosis
potentially enhancing phagocytic uptake by adjacent cells (77,101,102).
Lipocortin 1 was shown to protect cultured rat thymocytes from H2O2-elicited
necrosis. Glucocorticoid treatment, which induced lipocortin 1, stimulated
apoptosis while treatment with an anti-lipocortin 1 antibody enhanced necrosis
(103).
Glucocorticoids also mediate inflammation through interactions with the nuclear
factor kappa B (NF- kB) and
activator protein-1 (AP-1) signal transduction pathways (104-106). Both of these
pathways play a major role in the inflammatory and mitogenic responses in many
cell types and in general protect cells from apoptosis when stimulated by
cytokines, such as tumor necrosis factor
a (TNFa)
or oxidative stress (107-110). The NF-kB
transcription factor complex is usually retained in the cell cytosol in an
unstimulated state by an inhibitory protein (named Ik
B), which binds to the cytoplasmic NF-k
B complex and inhibits its translocation into the nucleus. TNFa
and other cytokines stimulate the phosphorylation of IkB,
which targets the inhibitor protein for proteolytic degradation, which then
frees the NF-kB complex for
nuclear translocation (See Figure 2 before last paragraph). Glucocorticoids
induce the expression of IkB,
thereby down regulating NF-kB
activation, whereas insulin also stimulates IkB
phosphorylation (111-114). This suggests that whether or not a cell will
initiate apoptosis in response to TNFa
will depend on the insulin: glucocorticoid ratio in its interstitial
environment.
Activated glucocorticoid receptor complexes are also able to inhibit
inflammatory and mitogenic transcription factors by direct protein-protein
interaction. These interactions have been demonstrated for the NFkB
complex, the AP-1 ligands c-fos and c-jun, and several STAT proteins, and are
independent of glucocorticoid-mediated transcription (106,115).
Despite their global anti-inflammatory effects, glucocorticoids have been shown
to potentiate certain aspects of the host defense system. For example, they have
been reported to induce expression of heat shock proteins such as HSP70 (116)
and increase activity of the DNA repair enzyme O6-methylguarnine-DNA
methyltransferase (117) in certain tissues. They also potentiate the effects of
interleukin-6 and hepatocyte-stimulating factor in inducing hepatic acute phase
proteins, such as Mn-superoxide dismutase and
a2-macroglobulin (118-122). Although both
glucocorticoids and lymphocyte stimulatory agents that are mediated via
intracellular Ca2+ or protein kinase c (e.g., calcium ionophors/phorbol esters,
antibodies to the T-cell antigen receptor) initiate apoptosis in maturing
lymphocytes, they are mutually antagonistic to the extent that glucocorticoids
protect lymphocytes from activation-induced apoptosis (123,124). Thus, the
effects of glucocorticoids on the inflammatory and immune systems are modulatory
rather than simply suppressive.
Inflammation, necrosis, oxidative damage and regenerative hyperplasia all play a
significant role in chemically induced tumor promotion, and glucocorticoids have
been shown to inhibit hyperplasia and neoplasia in a number of systems. For
example, glucocorticoids are used therapeutically as antineoplastic agents in
several types of leukemia and lymphoma (52,125), and they suppress growth of
certain lung or mammary adenocarcinomas (80,126-128). Dexamethasone has been
reported to inhibit both peroxisome proliferator-induced and lead
nitrate-induced proliferative hyperplasia in rat liver (129,130).
Glucocorticoids have also been shown to induce connexin expression and stimulate
gap junction formation in cultured hepatocytes and embryonic cells (131-133).
Inflammatory agents, such as phorbol esters, promote, and glucocorticoids
inhibit papilloma formation in mouse skin (134).
Toxic Effects of Glucocorticoids
Chronic and excessive elevation of glucocorticoid levels increases the risk
of developing hypertension, hyperkalemia, diabetes, atherosclerosis,
osteoporosis, glaucoma, and impairment of the immune and reproductive systems
(135,136). The organ most susceptible to glucocorticoid toxicity appears to be
the hippocampus. High doses of corticosterone administered to adrenalectomized
rats resulted in neuronal atrophy in the hippocampus, but not in other areas of
the brain (54,137,138). Because the hippocampus, in conjunction with the
hypothalamus, controls feedback regulation of the HPA, it was suggested by
Sapolsky and coworkers (138), in what has become known as the glucocorticoid
cascade hypothesis, that glucocorticoid-evoked hippocampal damage impairs the
feedback regulation of adrenal glucocorticoid output, which could result in
further increases in glucocorticoid levels and additional hippocampal damage.
Over a lifetime, such an effect may result in premature aging of the brain.
Evidence supporting this hypothesis includes
in vitro studies, which have demonstrated that glucocorticoids impair the
ability of cultured hippocampal cells to withstand neurotoxic stresses (138).
The proposed mechanisms responsible for these effects include inhibition of
glucose transport and disruption of Ca2+ homeostasis (138-140). In
humans, patients with Cushing's syndrome have been reported to exhibit memory
impairment, which correlated with serum cortisol levels (141), and dexamethasone
treatment has been reported to impair declarative memory performance (142).
However, although hypercorticism is often manifested in Alzheimer's patients
(139), long-term treatment with glucocorticoids is associated with delay in the
onset of Alzheimer's disease (143). Lipocortin 1 is expressed throughout the
brain, including the hippocampus, and has been shown to protect against neuronal
damage resulting from either ischemia or NMDA receptor agonists (144,145).
Exposure of adult rats to stress, hypercorticism or glucocorticoid therapy
reduces reproductive hormone levels in both sexes (3). In males, for example,
glucocorticoids appear to inhibit LH-mediated testosterone synthesis by cultured
rat Leydig cells (146) and dexamethasone treatment decreases, while
adrenalectomy increases serum testosterone levels in vivo
(147,148). In females, glucocorticoids decrease FSH-stimulated aromatase
activity and estrogen production by ovarian granulosa cells (149), suppress
ovulation and inhibit ovarian prostaglandin metabolism (150). They also inhibit
the preovulatory pituitary LH surge in female rats (151) and estradiol- and
gonadotropin releasing hormone-induced LH production in cultured rat pituitary
cells (152). In male rats, glucocorticoids inhibit pituitary secretion of
prolactin (153), but not mean LH levels (154). However, CRF and stress inhibit
pituitary LH secretion in both sexes (155,156).
Glucocorticoid-Mediated Effects of Dietary Restriction
Dietary restriction not only evokes anti-inflammatory and antineoplastic
effects that are consistent with chronic hypercorticism, but also protects the
aging rodent against insulin resistant diabetes (29,157-159), impaired tissue
growth and regeneration (160,161), certain neurological impairments (162,163),
and reproductive senescence (164,165) [See Table 1 following this paragraph].
Although these latter effects appear at first sight to be inconsistent with
hypercorticism, on further analysis they appear to be the natural consequence of
the nutrient stress that is produced by caloric restriction under the conditions
used for most experimental paradigms.
Table 1. Major Effects of Caloric Restriction |
Direct Effects |
Aging-dependent Effects |
Blood Glucose -
unchanged or decreased |
Neoplasia - delayed |
Pancreas, insulin
secretion - decreased |
Nephropathy - delayed |
Serum corticosterone - increased |
Cardiopathy - delayed |
Plasma ACTH - decreased |
Hyperinsulinemia - decreased |
Body temperature - decreased |
Cognitive defects -
decreased |
Hepatic gluconeogenic enzymes - increased |
Reproductive
senescence - delayed |
Cardiac muscle, myosin V1 - decreased, myosin V3
- increased |
Antioxidant enzymes - increased |
Pulsatile growth hormone - inhibited |
DNA repair - increased |
Hepatic IGF1 synthesis - decreased |
Pulsatile growth hormone - maintained |
Hepatic sex - specific drug metabolism - decreased |
Hepatic IGF1 synthesis - increased |
Cell proliferation - decreased |
Hepatic sex-specific drug metabolism - increased |
Apoptosis - increased |
Cell proliferation - increased |
Blood - leukopenia |
Blood - leukopenia |
Inflammatory response - decreased |
Lipocortin production - increased |
12-lipoxygenase - decreased |
Male gonadal steroids - feminized |
Reproductive function - decreased |
HSP70 - increased |
Direct effects of caloric restriction are those occurring immediately
after restriction is initiated and result directly from the organism's
response to caloric deficit. Aging-dependent effects of caloric restriction
are those occurring in response to the delay in physiological aging that
results from caloric restriction. Effects in italics are those which are
consistent with hypercorticism. References given in text or in references
(3, 21, 266).
|
There are several factors that differentiate the nutrient stress produced by
dietary restriction from other stress situations or glucocorticoid therapy (3).
Firstly, unlike treatment with pharmacological doses of synthetic
glucocorticoids, hypercorticism resulting from nutrient stress involves the
natural glucocorticoids, corticosterone or cortisol. The effects of these
natural glucocorticoids are mediated by serum transcortin and 11ß-hydroxysteroid
dehydrogenase, which may protect tissues from extreme hypercorticism (3).
Furthermore, unlike synthetic steroids such as dexamethasone, corticosterone and
cortisol bind to both Type I and Type II glucocorticoid receptors so that the
Type I receptor response is not inhibited concurrently with an excessive Type II
receptor response (166).
Secondly, the hypercorticism exhibited by dietary restricted rodents differs
from the continuously elevated serum corticosterone levels exhibited by starved
or chronically stressed rodents in that corticosterone levels are increased,
above those of their ad libitum-fed counterparts, only during a limited
circadian period that is prior to and coincident with feeding activity (167).
This type of intermittent hypercorticism appears to be less damaging to
mitogenic processes than continuously elevated glucocorticoid levels (3).
Thirdly, because the hypercorticism is a response to caloric deficit and
potential hypoglycemia and occurs in conjunction with normal feedback regulatory
systems, it is not associated with chronic hyperglycemia or hyperinsulinemia
(21,29,168). Thus, insulin resistance and protein glycation, which are the usual
pathological consequences of glucocorticoid-induced hyperglycemia, should not
occur. Instead, rates of intracellular glycation and oxidation of protein would
be expected to decrease in peripheral tissues due to reduced glucose
incorporation. Reduced collagen glycoxidation has been observed in skin from
calorically restricted rats (169), and accumulative oxidative damage to both
protein and DNA is reduced by dietary restriction in a number of tissues
(59,170-176).
Fourthly, under the usual conditions that are used for dietary restriction
experiments, significant hypercorticism only occurs during the early stages of
restricted feeding (25,28). In most strains of rodents used in caloric
restriction experiments, body weight gain is reduced in the restricted animals
to an extent where the body weight difference between the restricted and
ad libitum-fed animals equals or exceeds the caloric deficit (177) [See
Figure 3 following this paragraph]. Thus, during the latter half of a
calorically restricted rat's life span its caloric consumption per gram body
weight is equal to or greater than that of its ad libitum-fed
counterpart. Under these conditions significant hypercorticism would not be
required to protect the animal from potential hypoglycemia. As a consequence,
during senescence, when rodents are most susceptible to tissue degeneration due
to reduced capacity for cellular proliferation and reduced output of mitogenic
hormones (161,178); serum corticosterone levels are normally no longer
significantly increased in chronically calorically restricted animals (3,25,28).
Figure 3. Influence of Body Weight on Caloric Consumption in
Calorically Restricted Fischer 344 Rats
Fischer 344 rats, housed in a specific pathogen free barrier
facility at NCTR, were placed on a vitamin fortified NIH-31 diet at 60% of
ad libitum food consumption as described by Duffy et al (177).
A, the weight curves for male and female rats. B,
relative food consumption (expressed as food consumed per gram body weight by
the calorically restricted rats as a percentage of that consumed per gram body
weight by the ad libitum-fed rats) as a function of age. By 50 weeks
for the males and 70 weeks for the females, the calorically restricted rats
consume equivalent amounts of food per gram body weight as their ad libitum-fed
counterparts.
The effects of dietary restriction on biomarkers of mitogenesis are generally
consistent with the occurrence of hypercorticism during the early, but not the
late stages of caloric restriction. For example, caloric restriction from 16
weeks of age abolishes growth hormone pulsatility in six month-old male Brown
Norway rats, but pulsatility is restored in older animals (179). In male rats,
pulsatile growth hormone controls hepatic expression of both IGF1 and
sex-specific drug metabolizing enzymes such as cytochrome P450 2C11 (CYP2C11)
(180,181). As expected from its effects on pulsatile growth hormone, caloric
restriction decreases hepatic expression of both IGF1 and CYP2C11 in
young male rats (182,183). However, as the rats age, hepatic IGF1 and
CYP2C11 expression decreases in the ad libitum-fed rats, but is
maintained by caloric restriction animals so that in old rats hepatic IGF1
and CYP2C11 expression is greater in the calorically restricted animals
(182,183). This age-dependent biphasic effect of caloric restriction is
illustrated in Figure 4 (following this paragraph) and is a common feature of
several of the reported effects of caloric restriction in rodents. These
include: cell proliferation rates in kidney, pancreas and possibly liver from
B6D2F1 mice (161), serum DHEA levels in Fischer 344 rats (184), and
reproductive function in both rats and mice.
Figure 4. Age-dependent Effects of Caloric Restriction
(A) Schematic representation of the effects of caloric
restriction on mitogenic endpoints and reproductive function. Reduction in the
early burst of activity delays the degradation of these systems in old age.
Examples include: [BRDU], cell proliferation in kidney tubule cells from B6D2F1
mice, as measured by in vivo labeling with BrdU (161); [CYP] expression
of hepatic cytochrome P450 2C11 (CYP2C11) and its dependent activity,
testosterone 16a-hydroxylase in male Fischer 344 rats
(183); [IGF] expression of hepatic IGF1 mRNA in male Fischer 344 rats (182).
Caloric restriction decreases these parameters in young rats (B)
but maintains them in old rats (C).
The effects of caloric restriction on female reproductive function include
delayed puberty (185,186), inhibition of LH pulsatility concurrent with
hypercorticism (26), inhibition of ovulation (187), decreased litter size
(188,189), increased lactational diestrus (190), and reduced milk production
(191) during the initial period of caloric restriction and delayed reproductive
senescence during the later stage (165,188). In males, the initial effects of
caloric restriction include decreased LH pulsatility (192), reduced ratios of
serum testosterone to estradiol (193), decreased sperm motility in rats
(194,195), and decreased prostate weight, testicular sperm density and fertility
in mice (189). Long-term caloric restriction reduces testicular hyperplasia and
delays Leydig cell adenoma formation in old male rats (193,196), whereas chronic
feeding of a high caloric diet reduced reproductive performance in old male CF-1
mice (197).
The anti-inflammatory effects of caloric restriction are also generally
consistent with effects resulting from hypercorticism. For example, caloric
restriction has been reported to induce lipocortin 1 immunoreactive proteins in
rat liver (21), to inhibit carrageenan-induced inflammation in mice (30), to
decrease 12-lipooxygenase activity in rat liver and testes (3), to delay the
onset of autoimmunity in autoimmune-prone mice (198), and to inhibit promotion
of mouse skin papillomas by phorbol esters (199,200). In the last case,
adrenalectomy reversed the effect of caloric restriction whereas the effect was
enhanced by glucocorticoid replacement (11). Interestingly, caloric restriction
both potentiates regenerative hepatocyte proliferation in partially
hepatectomized rats (201) and reduces cell proliferation while stimulating
apoptosis in preneoplastic liver (202,203). Such an effect is consistent with
the reported dual synergistic and antagonistic effects of glucocorticoids on
TGFß in neoplastic and non-neoplastic hepatocytes (3,66). Dietary restriction
also reduces lung inflammation in rats exposed to ozone (204,205) and enhances
resistance to gram-positive bacteria, while lowering the production of
proinflammatory mediators elicited by endotoxin, a component of gram-negative
bacteria.
While old dietary restricted mice exhibited improved cognitive function, motor
performance, and reduced oxidative damage in the brain (162,163), dietary
restriction neither inhibited hippocampal aging in rats, nor appeared to be
overtly detrimental to the hippocampus (140,206). However, dieting and dietary
restriction have been reported to impair cognitive function in humans (207).
Despite potential endangerment to the hippocampus, hypercorticism during
nutrient stress would be expected to be beneficial, since the alternative,
hypoglycemia in conjunction with increased inflammatory activity, would pose a
greater threat to the entire central nervous system.
Taken together, dietary restriction in rodents appears to produce a series of
pleiotropic biochemical and physiological effects that are consistent with a
condition hypercorticism that is more severe in the early stages of caloric
restriction than in the later stages and that occurs without concurrent
hyperglycemia. The overall effect of this condition is to conserve energy by
minimizing metabolism, proliferation and nonessential functions in peripheral
tissues. This in turn appears to minimize damage to the affected tissues so that
the progression of degenerative or neoplastic lesions is delayed.
Dietary Restriction, Hypercorticism and Chemical Toxicity
Dietary restriction has been reported to increase the maximum tolerated dose
or LD50 of a number of chemicals (208,209) and to cause isoform-selective
alterations in drug-metabolizing enzyme expression (183). While relatively large
changes occur in sex-specific isoforms that are regulated by growth hormone,
other isoforms are either unaffected or show moderate, circadian-dependent
alterations (183). For example, 40% caloric restriction increased hepatic
CYP1A-selective 7-ethoxyresorufin O-deethylase and CYP2B-selective
7-pentoxyresorufin O-dealkylase activities and immunoreactive protein in both
male and female 18 week-old Fischer 344 rats, but only at specific circadian
time-points (183,210). Conversely, caloric restriction decreased and eliminated
the circadian variation of testicular CYP2A1-dependent testosterone 7a--hydroxylase
activity (193). In addition to altering drug metabolism, it is probable that
caloric restriction may also stimulate the renal clearance of drugs since
caloric restriction or fasting may cause polydipsia and increase diuresis and
natriuresis consistent with elevated ANP levels (177,211-213). It is possible
that both these effects of fasting on serum ANP levels and of caloric
restriction on polydipsia and hepatic CYP1A and CYP2B expression result from
hypercorticism, since glucocorticoids induce ANP levels (214), cause polydipsia
(215), and stimulate induction of CYP1A1 and CYP2B isoforms (216-218). In a
recent study (219), caloric restriction was shown to enhance the induction of
hepatic peroxisomal marker enzymes in B6C3F1 mice treated with chloral hydrate.
This was also consistent with restriction-induced hypercorticism because
glucocorticoids induce the hepatic peroxisome proliferator activated receptor
PPARa.
In several cases, the effects of dietary restriction on the metabolic activation
of genotoxic chemicals have been shown to correlate with specific isoform
expression. For example, in vivo and in vitro binding of
aflatoxin B1 to DNA was decreased in liver from caloric restricted
rats concurrently with decreased CYP2C11, whereas binding of benzo(a)pyrene to
DNA was increased concurrently with increased 7-ethoxyresorufin O-deethylase
activity (183,220,221). Dietary restriction has also been reported to reduce
endogenous DNA damage in liver, mammary gland and other tissues (59,170,171).
However, although there are several reports demonstrating that caloric
restriction increases DNA repair activity in a number of cell systems, the
effect is confined mostly to old animals (222).
Dietary restriction has been shown to reduce the severity or delay the onset of
carcinogenesis in rodents exposed to a number of chemical carcinogens including
aflatoxin B1, polycyclic aromatic hydrocarbons (PAH) and nitosamines
(220,223-226). Although dietary restriction clearly alters the initiation stage
of chemical carcinogenesis (220), it is now apparent that the major beneficial
effects of caloric restriction are associated with the promotion and progression
stages. This is best illustrated by experiments involving neonatal exposure of
male mice to PAH (227). When mice were injected (ip) with 6-nitrochysene at 8
and 15 days post partum, they exhibited a 100% incidence liver adenomas and
carcinomas when necropsied at 12 months of age. Dietary restriction (40%),
initiated at 14 weeks of age, completely inhibited liver tumor formation even
though the restriction was not started until after the initiation and early
promotion stages of the carcinogenesis process were complete (227). Such effects
are consistent with the anti-mitotic and anti-proliferative effects of caloric
restriction and hypercorticism that are described above.
The observation that the body weight of rodents used in cancer bioassays
directly correlates with terminal incidence of background tumors (228-231) is
also consistent with effects on growth and cell proliferation, playing a major
role in mediating the antineoplastic effects of caloric restriction. These body
weight-tumor correlations were demonstrated from analysis of the control animals
from cancer bioassays conducted by the National Toxicology Program (NTP). In
B6C3F1 mice, terminal lung tumor incidence exhibited a positive
correlation with body weight at nine months on test. Conversely, terminal liver
tumor incidence correlated optimally with body weight at 12 months on test
(229,232). A typical correlation graph for liver tumors in male mice is shown in
Figure 5 following this paragraph. In Fischer 344 rats, terminal pituitary tumor
incidence exhibited a positive correlation with body weight at 13 months on
test, whereas terminal leukemia incidence exhibited a positive correlation with
body weight at 14 weeks (233). Interestingly, caloric restriction initiated at
six weeks of age inhibited leukemia to a much greater extent than restriction
initiated at 14 weeks, whereas pituitary adenoma formation was affected equally
by both caloric restriction paradigms (233). This suggests that critical periods
exist when rodents are most susceptible to subsequent development of specific
cancer endpoints. This effect can also be demonstrated for background liver
tumors in B6C3F1 mice (231).
Figure 5. Association between Mean Body Weight and Liver
Neoplasm Incidence in Male B6C3F1 Mice
Data from control groups from nine NTP studies conducted during the
1980s and early 1990s that used water-based gavage for dosing. The percent liver
neoplasm values are the survival-adjusted rates of hepatocellular adenoma or
carcinoma. The individual studies used are listed in (251).
It would appear, therefore, that the rate of growth during the early adult
period of an organism's life determines its subsequent susceptibility to
neoplastic or degenerative diseases, and rates of growth are in part dependent
on glucocorticoid status and caloric intake. Glucocorticoids are a major
component of the stress and inflammatory responses, where their primary
functions appear to be: [1] to globally reduce energy consumption so that energy
may be channeled to the site of trauma or inflammation, and [2] to prevent
excessive tissue damage due to overexpression of the inflammatory response (83).
During severe nutrient stress, hypercorticism allows an organism to conserve
energy so that it may survive, but in the process, growth and reproductive
immune and cognitive functions may be compromised. However caloric excess may be
equally detrimental resulting in overstimulated growth, uncontrolled cell
proliferation, autoimmunity, inflammatory diseases, and neoplasia. Between these
two extremes lies a physiological window where health and longevity is
maximized. Hypercorticism, as a hormonal response to nutrient stress, appears to
be common to most mammalian species and most probably evolved as a mechanism to
ensure survival of the species through periods of famine (234-237). In times of
abundant food supply, rapid growth and fecundity are favored over endurance and
longevity. Conversely, when food becomes scarce reproductive performance and
growth are sacrificed in favor of extended total and reproductive lifespans,
thus increasing the probability that sufficient individuals will survive to
restore the population when conditions improve. This phenomena can be observed
in the human population, where rising living standards are correlated with
obesity and increased cancer rates (4), as well as in rodent strains that are
used in toxicology testing.
Consequences for Chronic Toxicity Testing
Over the last three decades, improvements in diet formulations and animal
husbandry techniques and commercial breeding considerations have resulted in a
general drift towards heavier animals for all the major rodent strains used in
toxicity testing (13,14,229,230). Increase in body weight in these strains is
frequently associated with decreased survival and increased susceptibility to
neoplastic and degenerative diseases (13,14,165). Furthermore, interlaboratory
variations in mean body weights and tumor incidence complicate comparison
between studies (18).
This effect can create problems for the interpretation of chronic cancer
bioassays. For example, the incidence of background liver tumors in control
B6C3F1 mice used for chronic bioassays, conducted by the NTP, has
been shown to vary between 5% and 75% (238). This increased variability is
partly due to altered housing conditions, but other factors such as genetic
drift may also be responsible (238,239). However, differences in mean body
weights between treatment groups within individual studies pose a greater
problem since they may result in artifactual assumptions about the
carcinogenicity of certain test chemicals (228,229).
Such differences usually arise when toxic responses to the test chemical reduce
body weight gain, and a 10% reduction in body weight gain has been used as a
criteria for achieving a maximum tolerated dose (240). Chemically induced body
weight reductions can arise for a number of reasons, including decreased food
consumption due to palatability problems in feed studies, anorexia due to toxic
stress, disrupted intestinal absorption, or toxic wasting syndromes due to
disruption of metabolism or endocrine systems. In most cases, such body weight
gain decreases would be expected to be associated with hypercorticism, which
would result from either nutrient or classic, CRF-mediated toxic stress.
Nutrient stress resulting from reduced food consumption would be expected to
decrease the inflammatory response in a similar manner to glucocorticoid
administration or caloric restriction, whereas stress due to chemical toxicity
would be expected to enhance the inflammatory response due to increased CRF and
interleukin levels (See Figure 6 following this paragraph). In addition,
excessive body weight gain in rodents may also involve an altered inflammatory
response. Such animals could exhibit reduced efficiency in leptin expression or
function, analogous to the ob/ob mouse, and this would result in hypothermia,
hypercorticism, hyperglycemia, but a generally reduced inflammatory response due
to elevated corticosterone (37,241,242). Conversely, they could exhibit
excessive food consumption, which would result in low corticosterone levels,
excessive production of arachidonic acid and an increased inflammatory response
(243). Excessive inflammation exacerbates toxic responses to chemicals
(244-246), directly promotes neoplasia in certain systems such as mouse skin
(134) and can result in degenerative conditions such as renal inflammatory
disease (243). Therefore, whether alterations in weight gain in bioassay rodents
are accompanied by changes in inflammatory response may influence the
relationship between body weight and terminal tumor incidence as illustrated in
Figure 6 (right after this paragraph). Such effects are not only relevant to
two-year cancer bioassays, but also to the ancillary studies associated with
these bioassays. As noted in the previous section, diet and body weight can
influence the toxicokinetics of many chemicals. Dietary restriction has also
been shown to influence rates of tumor progression in transgenic mouse models
that are currently being introduced for rapid carcinogenesis screens (247,248).
This should be considered during the interpretation of cancer bioassay data.
Figure 6. Factors Affecting the Relationship Between Body
Weight and Pathological Endpoints in Chronic Cancer Bioassays
The development of pathological lesions in rodents used in
chronic bioassays is influenced by both mitogenic and inflammatory effects.
Conditions, which increase inflammation in addition to increasing mitogenesis,
would be expected to increase the tumor risk to a greater extent than predicted
by body weight alone.
Dietary restriction has been suggested as a possible means for eliminating
background tumors from the bioassay control populations (14,15). However, as
stated above, dietary restriction inhibits chemically induced carcinogenesis in
rodents (230,238,249). Moreover, dietary restriction is generally implemented by
limiting food consumption to a set percentage of ad libitum food
consumption, and this may vary between rodent populations in different
laboratories (230,238).
An alternative approach involves using dietary control to manipulate the body
weights and growth rates of rodents used in bioassays so that they conform to
strain-specific standardized weight curves. Such standardized or idealized
weight curves have been created for male and female B6C3F1 mice and
could potentially be used throughout industry and the regulatory community to
standardize background neoplasm incidences between laboratories (3). The body
weights of mice used for both control and treatment groups in future bioassays
could be manipulated to fit these growth curves by moderate feed restriction or
dietary supplementation.
Testing Dietary Control
The concept of using idealized weight curves has recently been tested as part
of a standard NTP bioassay of chloral hydrate in B6C3F1 mice that was
conducted at the National Center for Toxicological Research [NCTR]
(219,231,250-252). Data from mice used in NTP and NCTR chronic bioassays and
aging studies were used to construct idealized weight curves for male and female
B6C3F1 mice that predicted a liver neoplasm incidence of 15% to 20%
at 26 months of age. A 15% to 20% liver neoplasm incidence is sufficiently high
to guarantee that the sensitivity of the mouse to chemical carcinogenesis has
not been compromised, and it is low enough to ensure that the spontaneous
neoplasms will not obscure any chemically induced liver tumors, and that
sufficient mice will survive to the end of a two-year study. Initially the
relationship between body weight and liver tumor incidence was calculated for
historical control populations of male and female ad libitum-fed mice
(approx. 2,750 and 2,300 animals respectively). However, it was determined that
male B6C3F1 mice, which had been subjected to forced body weight
reduction due to either dietary restriction or exposure to non-carcinogenic test
chemicals, differed from ad libitum-fed mice in their relationship
between body weight and tumor incidence. A second weight-reduced
historical control population (approx. 1,600 animals) was therefore used to
construct the idealized weight curve for male mice (231). These curves are shown
in Figure 7 following this paragraph.
Figure 7. Idealized Body Weight Curves for Male and Female
B6C3F1 Mice
Body weights for ad libitum-fed and 40% calorically
restricted mice from caloric restriction studies performed at NCTR are shown for
comparison. Food consumption data from these mice were used to construct a
feeding schedule used for manipulating the animals’ weights to fit the idealized
body weight curve. Taken from Leakey et al. (231), full details are
given in this reference.
Weight-reduced mice exhibited a more linear relationship between
body weight and liver tumor incidence in the low weight range than did ad
libitum-fed mice, which exhibited a J-curve profile (3,229,231). These
differences did not occur in females and were less apparent in sexually
senescent males older than 60 weeks (See Figure 8 following this paragraph) and
result in a larger sex-difference in liver tumor incidence in light mice than in
heavy mice (231).
Figure 8. Liver Tumor Risk Curves for Ad Libitum-fed
Male and Female, and Weight-Reduced Male B6C3F1 Mice at Various Ages
Liver tumor risk curves were constructed as described in Leakey et al
(231) from body weight values corresponding to the ages shown on each graph.
They are plotted as spline curves rather than bar graphs. The dotted line
represents the target tumor risk (17.5%) for the idealized weight curve.
It is probable that stress due to dietary restriction or chemical exposure
reduces not only the body weight-related liver tumors in male mice, but also the
sex-dependent liver tumors which occur independently of body weight in small
male B6C3F1 mice and cause the J-curve profile in the tumor risk
curve. Castration studies with the parent strains of B6C3F1 mice,
which also show sex differences in liver neoplasm risk, suggest that this
increased incidence of liver neoplasms in the small male mice is partly due to
testicular androgens (253-255). As discussed above, short-term caloric
restriction has been reported to reduce the testosterone/estradiol ratios and
impair male reproductive function in rodents, and restraint stress or food
depression suppresses LH secretion in male mice (256).
The NCTR bioassay of chloral hydrate compared dietary-controlled mice with
ad libitum-fed mice. Groups of 120 male mice received chloral hydrate in
distilled water by gavage at doses of 0, 25, 50, or 100 mg/kg, 5 days per week
for 104 to 105 weeks; vehicle controls received distilled water only. Each dose
group was divided into two dietary groups of 60 mice. The ad libitum-fed
mice had feed (NIH-31 autoclaved pelleted diet, Purina Mills, Richmond, IN)
available ad libitum, and the dietary-controlled mice received the same
feed in measured daily amounts calculated to maintain body weight on a
previously computed idealized body weight curve. Twelve mice from each diet/dose
group were evaluated at 15 months. Weekly feed allocation values required to
control body weight in mice to conform to the idealized body weight curve were
calculated as grams of NIH-31 pellets per day from food consumption and body
weight data from previous NCTR studies using B6C3F1 mice. This is described in
detail elsewhere (231). It was anticipated that individual mice would exhibit
body weights that differed significantly from the idealized body weight curve at
certain times during their growth. These mice were identified on a weekly basis
and their food allocation adjusted in either 1.0 or 1.5 g increments to
manipulate the body weight back onto the idealized body curve.
While chloral hydrate was less potent than expected, it did produce a weak, but
statistically significant, hepatocarcinogenic response in both the ad
libitum-fed and the dietary-controlled mice (See Table 2 immediately
following).
Table 2. Liver Neoplasms in
Ad-Libitum-Fed and Dietary-Controlled Male Mice in the Two-Year Gavage
Study of Chloral Hydrate |
|
Vehicle
Control |
25 mg/kg |
50 mg/kg |
100 mg/kg |
Hepatocellular Adenoma |
Ad Libitum-Fed |
Overall rate |
12/48 (25%) |
19/48 (40%) |
17/47 (36%) |
17/48 (35%) |
Adjusted rate |
25.2% |
40.8% |
37.8% |
36.2% |
Terminal rate |
9/41 (22%) |
14/37 (38%) |
15/36 (42%) |
16/44 (36%) |
First incidence (days) |
511 |
639 |
668 |
713 |
Poly-3 test (by dose) |
P=0.2362 |
P=0.0792 |
P=0.1373 |
P=0.1722 |
Dietary-Controlled |
Overall rate |
9/48 (19%) |
7/48 (15%) |
10/48 (21%) |
10/48 (21%) |
Adjusted rate |
19.1% |
15.2% |
21.2% |
21.8% |
Terminal rate |
9/45 (20%) |
7/44 (16%) |
10/47 (21%) |
9/41 (22%) |
First incidence (days) |
757 (T) |
757 (T) |
757 (T) |
625 |
Poly-3 test (by dose) |
P=0.3381 |
P=0.4111N |
P=0.5013 |
P=0.4753 |
Poly-3 test (comparison) |
P=0.3238 |
P=0.0046 |
P=0.0624 |
P=0.0951 |
Hepatocellular Carcinoma |
Ad Libitum-Fed |
|
|
|
|
Overall rate |
4/48 (8%) |
10/48 (21%) |
10/47 (21%) |
7/48 (15%) |
Adjusted rate |
8.5% |
21.4% |
22.0% |
14.7% |
Terminal rate |
2/41 (5%) |
5/37 (14%) |
5/36 (14%) |
4/44 (9%) |
First incidence (days) |
689 |
666 |
668 |
629 |
Poly-3 test (by dose) |
P=0.3737 |
P=0.0716 |
P=0.0631 |
P=0.2713 |
Dietary-Controlled |
Overall rate |
2/48 (4%) |
5/48 (10%) |
4/48 (8%) |
8/48 (17%) |
Adjusted rate |
4.2% |
10.9% |
8.5% |
17.3% |
Terminal rate |
2/45 (4%) |
5/44 (11%) |
4/47 (9%) |
4/41 (10%) |
First incidence (days) |
757 (T) |
757 (T) |
757 (T) |
486 |
Poly-3 test (by dose) |
P=0.0371 |
P=0.2078 |
P=0.3382 |
P=0.0422 |
Poly-3 test (comparison) |
P=0.3356 |
P=0.1364 |
P=0.0617 |
P=0.4740N |
Hepatocellular Adenoma or Carcinoma |
Ad Libitum-Fed |
Overall rate |
16/48 (33%) |
25/48 (52%) |
23/47 (49%) |
22/48 (46%) |
Adjusted rate |
33.4% |
52.6% |
50.6% |
46.2% |
Terminal rate |
11/41 (27%) |
16/37 (43%) |
17/36 (47%) |
19/44 (43%) |
First incidence (days) |
511 |
639 |
668 |
629 |
Poly-3 test (by dose) |
| P=0.0437 |
P=0.0684 |
P=0.1430 |
Dietary-Controlled |
Overall rate |
11/48 (23%) |
11/48 (23%) |
14/48 (29%) |
18/48 (38%) |
Adjusted rate |
23.4% |
23.9% |
29.7% |
38.6% |
Terminal rate |
11/45 (24%) |
11/44 (25%) |
14/47 (30%) |
13/41 (32%) |
First incidence (days) |
757 (T) |
757 (T) |
757 (T) |
486 |
Poly-3 test (by dose) |
P=0.0450 |
P=0.5728 |
P=0.3231 |
P=0.0844 |
Poly-3 test (comparison) |
P=0.1976 |
P=0.0030 |
P=0.0309 |
P=0.2975 |
(T)=
Terminal sacrifice; Overall rate = Number of neoplasm-bearing animals/number
of animals with tissue examined microscopically; Adjusted rate = Poly-3
estimated neoplasm incidence after adjustment for intercurrent mortality;
Terminal rate = Observed incidence at terminal kill. Beneath the
dietary-controlled group incidence are the P values corresponding to pair
wise comparisons between the ad libitum-fed group and the corresponding dietary-controlled group.
The Poly-3 test accounts for the differential mortality in animals
that do not reach terminal sacrifice. A lower incidence in the
ad libitum-fed group is indicated by N. |
In the ad libitum-fed mice, this consisted of a significant increase
in combined hepatocellular adenoma and carcinoma incidence in the 25-mg/kg-dose
group with no further increase at higher doses. In the dietary-controlled mice,
the combined hepatocellular adenoma and carcinoma incidence increased from 23.4%
in the control group to 38.6% in the 100 mg/kg dose group, with a statistically
significant dose trend; this increase was due to a statistically significant
increase in hepatocellular carcinomas in the high dose group. Observed numbers
of liver tumors were less in all the dietary-controlled dose groups than in the
corresponding ad libitum-fed dose groups. Dietary control also
significantly increased survival in the control, 25 and 50 mg/kg dose groups and
decreased body weight variability in all groups. Dietary control reduced
individual body weight variation in all four-dose groups (See Figures 9 & 10
immediately following).
Figure 9. Growth Curves for Ad Libitum-Fed and
Dietary-Controlled from the Chloral Hydrate Study - Low Dose and Vehicle Control
Groups
The graphs show the standard deviation of each weekly mean body weight
values for the control and 25 mg/kg chloral hydrate dose groups respectively.
The idealized weight curve and the NCTR historical growth curve for male B6C3F1
mice are shown on each graph for reference. The arrow marks the time point at
which 12 mice were removed for the interim evaluation.
Figure 10. Growth Curves for Ad Libitum-Fed and
Dietary-Controlled from the Chloral Hydrate Study -Medium and High Dose Groups
The graphs show the standard deviation of each weekly mean body weight
values for the 50 and 100 mg/kg chloral hydrate dose groups respectively. Other
details are given in Figure 9.
This was associated with smaller variation in related parameters. For
example, a significant dose-response in liver per body weight values was
observed in dietary-controlled mice used for an interim evaluation in the study
(252), whereas a significant dose response was not observed in the
ad libitum-fed mice, which exhibited much greater individual variation (See
Table 3 following this paragraph).
Table 3. Liver-Weight-to-Body-Weight Ratios in Male Mice Evaluated at 15
Months in the Two-Year Study of Chloral Hydrate |
Ad Libitum-Fed |
Dietary-Controlled |
0 mg/kg |
25 mg/kg |
50 mg/kg |
100 mg/kg |
0 mg/kg |
25 mg/kg |
50 mg/kg |
100 mg/kg |
n |
12 |
12 |
12 |
12 |
12 |
12 |
12 |
12 |
Mean* |
47.08 |
49.96 |
40.87 |
51.11 |
35.63 |
37.46 |
38.31 |
39.55 |
SDb |
17.59 |
17.40 |
4.13 |
19.58 |
1.02 |
1.37 |
2.09 |
2.29 |
SEMc |
5.08 |
5.02 |
1.19 |
5.65 |
0.30 |
0.39 |
0.60 |
0.66 |
Tukey’s testd |
A |
A |
A |
A |
A |
AB |
BC |
C |
Dunnett’s teste |
|
0.0001 |
0.0394 |
0.0017 |
0.0000 |
a |
Ratios are given as mg
liver per g body weight. |
b |
Standard deviation |
c |
Standard error of the mean |
d |
Each diet group was
treated on a separate ANOVA, and diet/dose groups not sharing the same
letter are significantly different from each other (P<0.05). |
e |
Beneath the vehicle
control group is the P value associated with the trend analysis. Beneath
the dosed groups are the P values relative to the vehicle control group. |
|
The dietary control procedures were relatively easy to run in this study and
did not generate a large amount of extra labor once the feed allocation software
had been developed. Access to a feed pellet sorter and prior experience with
caloric restriction studies also facilitated diet preparation. Since the study
used gavage dosing, weekly weights were readily available. The procedure would
be potentially more expensive and complicated for studies, which dose via the
feed because these animals are generally not weighed every week, and the
variable amounts of feed required for dietary control would result in variable
dose levels. However, dose variation occurs in all feed studies since individual
animals consume different amounts of feed. Dietary control could in fact
standardize dosing to a more defined level if the required level of dietary
restriction is relatively high and each animal consumes its entire daily feed
allowance (231, 250).
During the course of this study, a procedure was developed to use the
historical control data from ad libitum-fed and
weight-reduced mice to calculate predicted background liver tumor rates for
individual mice based on their body weight values between 21 and 68-weeks of
age. Full details of this procedure are given elsewhere (231). Using this
procedure, it was possible to predict background liver tumor rates for each
experimental group in the chloral hydrate study as shown in Table 4 (following
this sentence)
Table 4. Body Weight Derived Predictions of Liver Tumor Incidence in
Dietary-Controlled and
Ad Libitum-Fed Male B6C3F1 Mice Administered Chloral Hydrate |
Dose |
0 mg/kg |
25 mg/kg |
50 mg/kg |
100 mg/kg |
Observed Rates - Dietary Control |
Overall rate |
22.9% |
22.9% |
29.2% |
37.5% |
Poly 3 Adjusted rate |
23.4% |
23.9% |
29.7% |
38.6% |
Number with tumors |
11/48 |
11/48 |
14/48 |
18/48 |
Predicted Rates - sorted for < 5% ad libitum |
Poly 3 - Overall rate |
22.4 ± 2.3% |
22.3 ± 3.0% |
23.1 ± 2.2% |
21.5 ± 2.3% |
Adjusted rate |
22.9 ± 2.3% |
23.3 ± 3.0% |
23.1 ± 2.2% |
22.8 ± 2.0% |
Number with tumors |
11/48 |
11/48 |
11/48 |
10/48 |
Zh Statistic |
0.229 |
0.226 |
2.98 |
8.030 |
Significance P = |
0.4094 |
0.4145 |
0.0014 |
<0.00001 |
Predicted Rates - sorted by body weight decrease |
Poly 3 - Overall rate |
23.2 ± 3.2% |
24.0 ± 3.2% |
22.7 ± 3.3% |
21.6 ± 3.7% |
Adjusted rate |
23.6 ± 3.2% |
25.1 ± 3.1% |
23.1 ± 3.3% |
23.0 ± 3.5% |
Number with tumors |
11/48 |
12/48 |
11/48 |
10/48 |
Ztr Statistic |
0.073 |
0.376 |
2.001 |
4.493 |
Significance P = |
0.4710 |
0.3534 |
0.0227 |
<0.00001 |
Observed Rates - Ad Libitum-Fed |
Overall rate |
33.3% |
52.1% |
48.9% |
45.8% |
Poly 3 Adjusted rate |
33.4% |
52.6% |
50.6% |
46.2% |
Number with tumors |
16/48 |
25/48 |
23/47 |
22/48 |
Predicted Rates - sorted by body weight decrease |
Poly 3 - Overall rate |
33.8 ± 8.9% |
33.7 ± 9.5% |
33.6 ± 9.6% |
33.1 ± 7.8% |
Adjusted rate |
34.5 ± 8.9% |
35.8 ± 9.6% |
35.9 ± 9.6% |
34.1 ± 7.9% |
Number with tumors |
16/48 |
16/48 |
16/48 |
16/48 |
Ztr Statistic |
0.176 |
1.751 |
1.524 |
1.542 |
Significance P = |
0.4303 |
0.0400 |
0.0637 |
0.0616 |
Tumor risk was assigned to each mouse for each week of
evaluation by specific sort criteria as described in (231). The Ztr
statistics describe comparisons between the predicted survival adjusted
background tumor rate and the observed survival adjusted rated. The
predicted rates refer here to background liver tumor incidence predicted by
the body weight profiles of the individual mice in each dose group. Thus,
significant differences between predicted and observed rates in the groups
receiving chloral hydrate imply a carcinogenic effect due to the chemical.
|
and Figure 11 (following this paragraph), this procedure was able to
accurately predict the background tumor rates in both the dietary-controlled and
the ad libitum-fed dose-control groups. As illustrated in Figure 11
(following this paragraph), the variation in predicted background liver tumor
risk of individual mice in each dose group was much less for the
dietary-controlled mice than for the ad libitum-fed mice. Furthermore,
the technique showed the observed liver tumor incidence in the
dietary-controlled 50 mg/kg and 100 mg/kg dose groups were significantly greater
than predicted background tumor incidence even though these groups did not show
a statistically significant increase on the Poly-3 test (Table 2). This is
because the Ztr statistic used in Table 4 (above this paragraph) is
an estimate of the probability that the observed tumor rate is an acceptable
background tumor rate for the body weight-adjusted historical control population
and is dependent on the variance of calculated tumor risk of the mice in each
group rather than assuming a fixed binomial variance (231). As such it can give
valuable supportive evidence on the relevance of test chemically induced
increases in tumor incidence, but it assumes that no other factors significantly
influence tumor incidence between studies other than body weight, survival and
the test chemical.
Figure 11. Cumulative Tumor Risk Plots for the Chloral Hydrate Study
The mice from each experimental group were sorted by tumor risk into sequential
2% incremental groups. Tumor risk was calculated for the ad libitum-fed
mice by sorting by body weight decrease and for the dietary controlled mice by
sorting by "< 5% ad libitum" (see Table 4). Each mouse is represented
in the incremental group by its a value to adjust for intercurrent
mortality. The Gaussian distributions for each experimental group are calculated
from the means and standard deviations of the adjusted tumor risk values given
in Table 4. Taken from Leakey et al (231), full details are given in
this reference.
Dietary control, therefore, can potentially improve both the sensitivity and
reproducibility of cancer bioassays in mice. However, mouse liver neoplasms are
frequently induced epigenetically by chemicals, which appear to not be
carcinogenic for humans (257-259). Moreover, although incidence of liver cancer
is increasing in the U.S. and other Western countries, the primary risk factor
appears to be chronic inflammation resulting from hepatitis C or B infection
rather than linked to the ongoing rise in obesity or exposure to chemical
carcinogens (260-262). It could therefore be argued that a more sensitive mouse
bioassay would merely compound the problem of accumulating misleading or false
positive animal data that are irrelevant to human risk. Thus, it might not be
useful to the regulatory community.
There are two main answers to this. First, it must be remembered that most
other neoplastic lesions are also reduced by caloric restriction and related
stress responses. For example, in B6C3F1 mice positive correlations
have been reported between body weight and incidence of tumors of the pituitary
gland, lung and Harderian gland and of hemangiomas/hemangiosarcomas in addition
to liver tumors (229,232). Caloric restriction has also been shown to delay or
inhibit the development of these tumor types in B6C3F1 mice (263).
Reducing variability and body weight artifacts will therefore increase
sensitivity to detect a wide range of neoplastic responses in addition to liver
tumors.
Second, many potent genotoxic chemicals also cause liver tumors in B6C3F1
mice, and several are hepatocarcinogenic in humans (264,265). Evidence as to
whether a positive tumor response has relevance to humans and whether safe
exposure levels can be determined depends on mechanistic data ancillary to
bioassay tumor data. The emerging revolution in “-omics “ technology holds
promise that such mechanistic data will become more comprehensive and
informative, but it is dependent on the quality and reproducibility of available
tissue samples. Microarray techniques are especially vulnerable to sample
variation, because of the large number of interactive endpoints that have to be
measured simultaneously. Dietary control offers an approach to greatly reduce
both variability within studies and between studies.
Acknowledgments
We thank Ronald Hart, Peter Duffy and Angelo Turturro for helpful
discussions; Clyde Ulmer, Barbara Hampton, Sandra Goldman, and Kathy Carrol of
Northrop Grumman, NCTR, for their help in data processing; Martin Nichols, The
Bionetics Corporation, NCTR, for his expertise in maintaining the mice on
dietary control; and Richard Morris and Analytical Sciences Inc., Durham, N.C.,
for providing data from the NTP database. This work was sponsored in part by
NIA-NCTR contract 224-86-001, NIEHS-NTP contract 224-933-0001 and the NCTR.
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Glossary
Apoptosis: Apoptosis is a process of programmed
cell death triggered by either external hormonal signals or internal damage,
which is characterized by ordered degradation of chromatin and the rapid
phagocytosis of cellular debris without the release of inflammatory mediators.
Thus, apoptosis differs from necrotic cell death in that there is no associated
inflammation or compensatory mitogenesis.
Body weight reduction: In general, body weight
reductions resulting from dietary restriction are reductions in body weight gain
relative to corresponding ad libitum -fed control animals. That is the
restricted animals are gaining weight but at a slower rate. However, individual
animals, particularly older rodents on long-term dietary restriction studies,
may actually lose weight on a short-term basis, but generally not on a long-term
basis unless the weight-loss is associated with morbidity.
Dietary restriction: The term is used loosely here
to incorporate both food restriction and caloric restriction. Caloric
restriction is defined as the balanced and moderate reduction of the
carbohydrate, protein and fat content of a diet without a reduction in vitamin
or micronutrient content. Caloric restriction by 10 -40% of ad libitum
food consumption has typically been used in aging studies. Food restriction,
where total food intake is reduced, is simpler to perform and has been used in
several toxicity studies. It runs the risk of causing vitamin deficiencies when
used in excess.
Dietary control: As used here, dietary control
involves the manipulation of food allocations to maintain experimental animals
on a pre-determined body weight growth curve. It can involve either dietary
restriction of dietary supplementation depending on the individual animal and on
the required growth curve.
Glucocorticoid: A steroid ligand of the
glucocorticoid receptor, which evokes the glucocorticoid effect of raising blood
glucose levels. Includes pharmaceuticals such as dexamethasone
Hypercorticism: Elevated secretion and blood
concentrations of adrenal corticosteroids; predominantly corticosterone in
rodents and cortisol in primates.
Nutrient stress: Hypoglycemia-induced stress
resulting from reduced caloric intake, which can result from dietary
restriction, short-term fasting or starvation.
Restraint stress: Experimental technique of
producing a classic stress response in rodents by restricting their movements.
Weight-reduced mice: Mice which had lower body
weight values and lower rates of growth due to either dietary restriction or
exposure to a non-carcinogenic test chemical.
See reference 231 for more details. See also legends of Figures 1 and 2
for full names of hormones and signal transduction proteins .
The Authors
Dr. Julian Leakey earned his B.Sc. and Ph.D. degrees in the Department
of Biochemistry at the University of Dundee, Scotland, U.K. He joined the
Division of Reproductive and Developmental Toxicology at NCTR as a senior staff
fellow in 1985. Between 1991 and 1997 he worked on the NCTR-NIA Project on
Caloric Restriction. He is currently a member of NCTR’s Office of Scientific
Coordination. He became a Diplomate of the American Board of Toxicology in 1995.
His research interests include regulation of expression of drug metabolizing
enzymes, effects of diet and obesity on inflammatory processes and
carcinogenesis, and development and standardization of new animal models for use
in chronic toxicity studies.
Dr. William Allaben is Associate Director for Scientific Coordination
and the U.S. Food and Drug Administration’s (FDA) Liaison to National Toxicology
Program (NTP), and as such directs the FDA’s participation in the NTP. Dr.
Allaben received his Ph.D. in Physiology/ Pharmacology from Southern Illinois
University and worked as a Research Fellow at the National Center for
Toxicological Research (NCTR) before joining NCTR as a researcher in the
Division of Carcinogenesis. Allaben is an Adjunct Professor in the Department of
Toxicology and Pharmacology, University of Arkansas for Medical Sciences, Little
Rock and an elected Fellow in the Academy of Toxicological Sciences. He has
received numerous FDA recognition and honor awards including the Commissioner’s
Special Citation/ Health Claims Task-force and the Award of Merit, FDA’s highest
honor award. Dr. Allaben, with expertise in toxicology and carcinogenesis, has
over 28 years experience conducting and/or managing regulatory research and
testing programs.
John E. Seng is a Research Scientist/Study Director at Charles River
Laboratories-Arkansas Division, Redfield, Arkansas. Dr. Seng received his Ph.D.
in Interdisciplinary Toxicology from the University of Arkansas Medical Sciences
in 1994 and worked as a National Toxicology Program Research Fellow (1994) and
Study Director/Staff Fellow (1999) until joining Charles River Laboratories in
1999. During his tenure at NCTR, Dr. Seng's research focused on the refinement
and standardization of new animal models for use in chronic toxicity studies and
the influence of diet and obesity on detoxication pathways in the male
reproductive system.
Regulatory Research Perspectives Editorial Board
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Joanne N. Locke – Office of the Commissioner (OC)
Edward E. Max, Ph.D. – Center for Biologics Evaluation and Research (CBER)
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(NIEHS)
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