Environmental Health Perspectives Volume
102, Supplement 11, December 1994
[Citation
in PubMed] [Related
Articles]
Applications of Physiologic Pharmacokinetic Modeling in Carcinogenic
Risk Assessment
Daniel Krewski,1,2 James R. Withey,1 Lung-fa
Ku,1 and Melvin E. Andersen3
1Health Protection Branch, Health and Welfare Canada, Ottawa,
Ontario, Canada; 2Department of Mathemetics and Statistics, Carleton
University, Ottawa, Ontario, Canada; 3Chemical Industry Institute
of Toxicology, Research Triangle Park, North Carolina
Abstract
The use of physiologically based pharmacokinetic (PBPK) models has been
proposed as a means of estimating the dose of the reactive metabolites of
carcinogenic xenobiotics reaching target tissues, thereby affording an opportunity
to base estimates of potential cancer risk on tissue dose rather than external
levels of exposure. In this article, we demonstrate how a PBPK model can
be constructed by specifying mass-balance equations for each physiological
compartment included in the model. In general, this leads to a system of
nonlinear partial differential equations with which to characterize the
compartmental system. These equations then can be solved numerically to
determine the concentration of metabolites in each compartment as functions
of time. In the special case of a linear pharmacokinetic system, we present
simple closed-form expressions for the area under the concentration-time
curves (AUC) in individual tissue compartments. A general relationship between
the AUC in blood and other tissue compartments is also established. These
results are of use in identifying those parameters in the models that characterize
the integrated tissue dose, and which should therefore be the primary focus
of sensitivity analyses. Applications of PBPK modeling for purposes of tissue
dosimetry are reviewed, including models developed for methylene chloride,
ethylene oxide, 1,4-dioxane, 1-nitropyrene, as well as polychlorinated biphenyls,
dioxins, and furans. Special considerations in PBPK modeling related to
aging, topical absorption, pregnancy, and mixed exposures are discussed.
The linkage between pharmacokinetic models used for tissue dosimetry and
pharmacodynamic models for neoplastic transformation of stem cells in the
target tissue is explored. -- Environ Health Perspect 102(Suppl 11):000-000
(1994)
Key words: pharmacokinetic model, pharmacodynamic model, tissue
dosimetry, carcinogenic risk assessment
This article was presented at the Workshop on Pharmacokinetics:
Defining the Dose for Risk Assessment held 4-5 March 1992 at the National
Academy of Sciences in Washington, DC.
Address correspondence to Daniel Krewski, Health Protection
Branch, Health and Welfare Canada, Ottawa, Ontario, Canada K1A OL2. Telephone
(613) 954-0164. Fax (613) 952-9798.
Introduction
Pharmacokinetics is the study of the absorption, distribution, metabolism,
and elimination of xenobiotic agents in biologic systems. By studying the
fate of xenobiotics upon entering the body, it is possible to obtain information
on the amount of both the parent compound and reactive metabolites reaching
tissues that may be targets for the induction of cancerous lesions. This
affords an opportunity to incorporate data on tissue dose into pharmacodynamic
models of carcinogenesis.
Considerable experience has now accumulated with the development of physiologically
based pharmacokinetic (PBPK) models to describe the disposition of carcinogenic
xenobiotics. Each compartment in a PBPK model represents a physiologically
defined component of the body, such as blood or specific organs and tissues.
Physiologic parameters such as body weight, blood flow rates, and tissue
volumes are used to characterize the distribution of xenobiotics within
the structure provided by the PBPK model; biochemical parameters such as
partition coefficients govern uptake within target tissues. Metabolism in
the liver or other tissues is generally described by first-order or Michaelis-Menten
kinetics. PBPK models capable of accurately describing chemical disposition
within mammalian systems have been developed for at least 15 chemical substances
(1).
Traditional methods for predicting potential carcinogenic risk in humans
from animal data involve a number of empirical assumptions (2). First,
for low levels of exposure, it is generally assumed that carcinogenic risk
is directly proportional to the level of exposure. Second, it is assumed
that humans are at least as sensitive as the most sensitive animal species.
In addition, interspecies scaling of carcinogenic potency is done in relation
to body surface area or body weight, or an intermediate scaling factor (3,4).
And third, it is tacitly assumed that the dose of the proximate carcinogen
reaching the target tissue is proportional to the level of exposure to the
parent compound. It should be noted, however, that classic pharmacokinetic
and biochemical studies have indicated that some metabolic pathways are
saturable, even at low levels of exposure. This phenomenon could give rise
to a nonlinear dose-response relationship with a biochemical threshold.
The comparatively recent application of PBPK modeling to predict the
dose of the proximate carcinogen delivered to the target tissue obviates
the need to rely on empirical assumptions in carcinogenic risk assessment.
In particular, PBPK models provide a basis for a more biologically based
approach to dose, route, and species extrapolation. While physiologic modeling
thus provides a more rational approach to risk assessment, there may be
a number of uncertainties associated with its use since a moderately large
number of parameters, each subject to some degree of error, are required
for risk assessment applications.
The purpose of this article is to review the collective experience to
date in developing PBPK models for carcinogenic chemicals, with a view to
evaluating their role as a tool for obtaining more accurate predictions
of carcinogenic risk through improved tissue dosimetry. We begin with a
step-by-step overview of the process of building a PBPK model, defined in
terms of mass-balance equations for individual compartments included in
the model ("Development of a PBPK Model"). Appropriate measures
of tissue dose are discussed in "Tissue Dosimetry." Because PBPK
models may involve in excess of 20 or more parameters, we review recent
investigations designed to identify those parameters to which predictions
of tissue dose are most sensitive ("Sensitivity Analysis"). The
use of PBPK models developed to describe the fate of methylene chloride,
styrene, ethylene oxide, 1,4-dioxane, 1-nitropyrene, as well as polychlorinated
biphenyls, dioxins, and furans is reviewed in "Applications of PBPK
Modeling." Special considerations in the application of PBPK models
are discussed in "Other Considerations in Applications of PBPK Models,"
including the effects of aging, topical adsorption in inhalation studies,
pregnancy, and exposure to complex mixtures. The linkage between pharmacokinetic
models used for tissue dosimetry and pharmacodynamic models of carcinogenesis
is explored in "Pharmacodynamics."
Development of a PBPK Model
A PBPK model envisages the body as being comprised of physiologically
similar compartments. Each compartment represents an organ or tissue group,
and linked to the central blood compartment by arterial and venous blood
flow. The model is characterized by physiologic parameters such as tissue
volumes and blood flow rates, biochemical parameters such as the partition
coefficients, and kinetic parameters for metabolism and removal. These parameters
are used to provide a mathematical description of the model using mass-balance
equations for individual compartments.
General Organ Compartment
Figure 1. Schematic
diagram of a general organ compartment: Ci(t) = concentration
of xenobiotic in tissue i at time t; Qi
= blood flow rate; Ui = tissue volume; A(t) = concentration
of xenobiotic in arterial blood; Vi(t) = concentration
of xenobiotic in venous blood; Yi(t) = amount of xenobiotic
directly entering compartment; Zi(t) = amount of xenobiotic
directly removed from compartment.
Consider first the general organ shown in Figure 1. A xenobiotic that
has been taken up by the body and entered the bloodstream may enter the
compartment through the arterial blood and leave through the venous blood.
In certain compartments, the compound may enter or leave the compartment
directly by nonarterial routes. For example, a compound could be removed
from the liver by metabolism or from the kidneys by excretion. Compartment
kinetics can be represented by the mass-balance equation:
Note that the concentration in the arterial blood is the same in all
compartments. The allometric parameters such as tissue volume and blood
flow rate involved in this equation are generally obtained by referring
to published reference values (5).
Many PBPK models regard tissue uptake of chemical to be flow limited
(6). It assumes that the venous blood from the tissue is in equilibrium
with chemical in the tissue. This condition is related to the partition
coefficient between tissue and blood. So
where Pi denotes the partition coefficient between
tissue and blood. The partition coefficient represents the ratio of the
concentration of the compound in the tissue relative to that in blood under
steady-state conditions.
For some compounds like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),
the ability of the tissue to retain the compound is limited by the availability
of specific binding media such as protein. In this event, the linear relationship
in Equation 2 must be replaced by a more complicated one allowing for saturation
of protein binding (7).
The term dYi(t)/dt represents
the rate at which the compound enters the compartment directly. This can
occur in blood or skin at time t0 with intravenous or
dermal exposure. Direct entry with a single exposure can be represented
by the Dirac delta function:
where d(t) = 1 for t = t0 and is
0 otherwise. Continuous infusion from time t1 to t2
is represented by a step function with
First-order absorption following a single exposure at time t =
0 is represented by:
where ka > 0 represents the kinetic rate coefficient
for absorption.
The term dZi(t)/dt represents the rate of
removal of compound directly from the compartment. This includes the excretion
of the compound from the body and the bioconversion of the substance into
other compounds by metabolism. Metabolism is usually modeled using either
linear kinetics
or saturable nonlinear kinetics
Here ki is a first-order kinetic rate coefficient that
can be estimated from metabolic studies. The parameter Vmax
is the maximum rate of metabolism under Michaelis-Menten kinetics, and Km
is an equilibrium constant that is numerically equal to the concentration
of the substrate when the rate of metabolism is one-half of Vmax.
At low dose where the venous blood concentration Vi is
less than Km, the Michaelis-Menten Equation 7 can be approximated
by a linear process as in Equation 6 with a rate coefficient of Vmax/Km.
At high doses, the Michaelis- Menten equation approximates a zero-order
process with dYi(t)/dt * Vmax.
The metabolism of methylene chloride involves both the linear (glutathione-S-transferase
[GST]), pathway and the saturable (mixed-function oxidases [MFO]) pathway
(10). Therefore, both removal processes (Equations 6 and 7) are required
in the PBPK model for this compound.
With benzene, the first-generation metabolite (benzene oxide) is further
metabolized into several phenol conjugates, phenyl mercapturic acid conjugates,
and hydroquinone conjugates. The generation of these secondary metabolites
is considered as a separate process outside of the original five-compartment
PBPK model for benzene (11).
Blood compartment
The mass-balance equation for the blood compartment takes a slightly
different form from that in Equation 1 to satisfy the conservation of mass
across all compartments. Specifically it is expressed as
where Qb=iQi,
with
i devoting the summation excluding the blood
compartment.
For most compounds, the concentration in arterial blood leaving the blood
compartment is the same as the blood concentration within the compartment.
However, for TCDD, it is necessary to allow for binding to blood proteins.
This effectively reduces the concentration in arterial blood flowing into
different compartments (7).
Blood-Lung Compartment
For a volatile compound, the function of the lung must be included to
represent the transfer of the compound between inhaled and exhaled air.
The compartmental diagram in Figure 1 is then modified as shown in Figure
2, and the mass-balance equation in Equation 8 becomes:
where Qalv is the alveolar ventilation rate, Cinh
is the concentration of the compound in inhaled air, and Calv
is the concentration of the compound in alveolar tissue. The alveolar ventilation
rate is easily obtained by direct measurement. The concentration of the
compound in inhaled air and alveolar tissue can be expressed as
and as:
respectively, where Ca(t) denotes concentration
in air, and Pa is air-blood partition coefficient.
Figure 2. Schematic
diagram of the blood-lung compartment.
A more complex representation of the blood-lung compartment has also
been used in the study of methylene chloride to take into account metabolism
in the lung, which is one of methylene chloride's target organs (10).
The model uses one compartment to represent the exchange of methylene chloride
between air and blood, and another to represent metabolism.
Selection of Physiologic Compartments
The body can be divided into two broad classes according to the value
of the ratio Ui/Qi in Equation 1. This ratio
represents the period of time required to replace the volume of blood present
in the ith compartment. The first class includes richly perfused tissues
such as the liver and kidney; the second includes poorly perfused tissue
such as skin and fat (13). Since metabolism in the liver represents
a special function, it is usually considered as a separate compartment.
Since fat will have much larger partition coefficient for lipophilic compounds,
it may be considered as a separate compartment when studying fat-soluble
substances. These considerations suggest the use of a PBPK model with the
following five compartments: blood, liver, richly perfused tissue, poorly
perfused tissue, and fat. For volatile compounds, the blood compartment
may be replaced by a blood-lung compartment. With some variations, this
basic five-compartment model has been widely used in PBPK modeling.
Solution of a PBPK Model
Consider the five-compartment PBPK model shown in Figure 3, in which
the compound enters the blood compartment directly and is metabolized principally
in the liver. The dynamics of this system are governed by the mass-balance
equations representing the function of each compartment. If all of these
equations are linear, then their solution can be expressed in closed form.
More generally, the solution of both linear and nonlinear models can now
be obtained using numeric methods. Computer programs such as Advance Continuous
Simulation Language (ACSL)(14) and SIMUSOLV (15) specifically
designed for this purpose are both efficient and easy to use.
Figure 3. Schematic
diagram of a five-compartment PBPK model.
To illustrate the solution of a PBPK model, consider the physiologic
data in Table 1 for Sprague-Dawley rats previously reported by Leung et
al. (7). Solutions to the five-compartment PBPK model shown in Figure
3 based on these values are illustrated in Figure 4 for the case of a single
iv bolus injection of the test compound. The concentrations in each of the
five compartments are shown in Figure 4A when there is no removal and all
partition coefficients are equal to unity. In this case, the blood concentration
is highest at the time of injection. As the compound is distributed to other
compartments, the concentration in the blood decreases. At steady state,
the concentration is the same in all compartments.
Figure 4. (A)
Concentration in different compartments with no removal: Pr=,
Pe=, Ps=, P=1; (B) Concentration
in different compartments with no removal: Pr=2, Pt=3,
Ps=5, Pf=10.
The richly perfused compartment has the smallest flushing time Ur/Qr.
Because the blood is being replaced quicker in this compartment than others,
the concentration of the compound in the richly perfused compartment increases
more rapidly than in other compartments. The concentration in this compartment
reaches its peak when the venous and arterial blood concentrations are equal.
Since the concentration in the blood compartment continually decreases because
of distribution to other compartments, eventually the concentration in the
richly perfused compartment surpasses that of the arterial blood. Consequently,
the richly perfused compartment starts to release the compound back into
the blood where it is transported to other compartments. Furthermore, since
the richly perfused compartment is the first compartment whose venous blood
concentration reaches that of the arterial blood, its peak concentration
will exceed that in other compartments.
The compartment with the next smallest flushing time is the liver, in
which the peak concentration is achieved after that of the richly perfused
compartment. Both slowly perfused and fat compartments have large blood
flushing times. Consequently, the concentration in these two compartments
increases slowly. The peak venous blood concentration occurs at a much later
time, and is smaller in amplitude. At steady state, the concentration in
all compartments reaches the same equilibrium level.
When different partition coefficients are assigned to different compartments,
the chemical flushing time UiPi/Qi
assumes the role of the blood flushing time. The partition coefficients
for blood and for richly perfused tissues are generally comparable, so that
there is no change in the order in which compartments achieve their peak
concentrations. However, the partition coefficient for fat can be much larger
than that for slowly perfused tissue, so that the peak concentration in
fatty tissue could occur at a much later time. The steady-state concentration
in different compartments are proportional to their respective partition
coefficients, as shown in Figure 4B.
The same model was modified to include saturable metabolism in the liver
with Km = 0.36 and Vmax = 3.6 (in arbitrary
units of measurement). Different doses 1, 10 and 100 are used to demonstrate
the effect of saturation of removal. Figure 5 shows the concentration of
the compound in the liver compartment, plotted on a semi-logarithmic scale.
At the lowest dose, the venous blood concentration in liver is below Km
at all times, so that removal follows linear kinetics. At the middle dose,
the venous blood concentration in the liver is above Km
near its peak. At this time, removal reaches its maximum rate and cannot
proceed at a faster rate. At all other times, removal is essentially linear.
Because of this saturation effect, the difference between the liver concentration
at the low and middle dose is not consistent over time. With saturation
of removal at the highest dose, more of the compound is retained in the
liver. The differences among the three curves are therefore largest near
their peaks.
Figure 5. Liver
concentration with saturable removal (Vmax = 3.6, Km
= 0.36.
Tissue Dosimetry
Some measure of the level of reactive metabolites reaching the target
tissue should provide a better dose metameter for risk assessment purposes
than the administered dose. However, consideration needs to be given to
the most appropriate way to express tissue dose.
Integrated Tissue Dose
Andersen (16) used an integrated measure of tissue dose given
by the area under the concentration-time curve (AUC) for either the parent
compound or its reactive metabolites in the tissue concerned. Specifically,
the AUC for the parent compound in blood is given by
[12]
For metabolites formed in the liver, we have
[13]
which is simply the total amount of metabolite formed divided by the
volume of the liver. The AUC in blood can be estimated directly by taking
blood samples at frequent time intervals. However, this cannot be done in
most other compartments, since tissue samples generally require destructive
invasive sampling. Once a PBPK model has been developed, however, it can
be used to predict tissue doses in any of the model compartments. Specific
target tissues, if they are known, may be incorporated into the model as
separate compartments.
For a linear PBPK model, the AUC of the parent compound in blood can
be expressed as
[14]
following administration of a single iv dose d of a xenobiotic.
Note that this expression involves only the blood flow rate Ql
to the liver and the rate of metabolism kl in the liver.
The AUCs of the parent compound in other compartments are related to those
in blood. Specifically, it can be shown that
Because of the relationship in Equation 2 between the concentration in
a tissue compartment and venous blood leaving the compartment, the AUC for
venous blood is the same for all compartments.
For a volatile compound with the blood compartment replaced by a blood/lung
compartment, we have
where G=1/(1+QalvPa/(1/Ql+1/kl)).
The same factor G is applied to all other integrated doses given
in Equations 15 to 19. This is similar to the steady-state solution for
the metabolic clearance fraction given by Bogen (17).
Equations 15 to 18 provide simple relationships between the AUC in blood
and other compartments. Since AUCb can be determined directly
from blood concentration measurements, these relationships could be used
to predict the AUC in other compartments without constructing a complete
PBPK model. Note that Equations 15 to 18 make no assumptions about the number
of compartments included in the model, or the nature of metabolic processes
(linear or saturable).
In a linear system, the only parameters which are important for the computation
of the integrated dose are the blood flow rate to the liver Ql,
the removal rate of the compound in the liver kl, and
the partition coefficient P for the specific compartment. Other physiologic
parameters such as tissue volume do not enter into the calculation. An increase
in compartmental volume will increase the blood flushing time (as discussed
previously) as well as delay the occurrence of the peak concentration and
reduce amplitude. However, the AUC in that compartment remains unchanged.
The relative magnitudes of the AUCs in different compartments are determined
solely by the partition coefficients. A change in the volume or blood flow
rate in a compartment will only change the shape of the concentration-time
curve, not the integrated tissue dose.
The integrated dose is dependent on the blood fiow rate into the liver
compartment. Because an increase in blood fiow into the liver will increase
the supply of the compound to liver tissue, and hence increase removal.
Since the blood concentration declines monotonically following administration
of a single iv dose, an increase in the rate of the change in the liver
concentration will decrease AUCl.
A compact solution for AUCb similar to that in Equation
14 cannot be found for a nonlinear PBPK model. However, the simple relationships
given in Equations 15 to 18 remain valid (18). Equation 20 now assumes
the more complex form
[21]
Here the integrand is the rate of metabolism, and the integral on the
left-hand side is the total amount of metabolite produced. This equation
simply refiects the fact that the total amount of the metabolite produced
should equal to the amount of the parent compound administered.
Sensitivity Analysis
From the systems analysis point of view, a PBPK model can be seen as
a system where the level of exposure to a xenobiotic represents the input,
and the dose of the reactive metabolite reaching the target tissue represents
the output. The output relates to the input by means of a PBPK system that
typically involves 20 or more parameters. Systems engineers often conduct
analyses to determine how sensitive the model outputs are to changes in
the values of the model parameters. Sensitivity analyses of this type offer
a means of estimating the uncertainty in predictions of tissue doses conferred
by uncertainty in the PBPK model parameters. Sensitivity analysis may also
identify critical parameters that contribute most to the overall level of
uncertainty in model outputs.
The flrst analysis of the uncertainty in PBPK model outputs was conducted
by Portier and Kaplan (19). These investigators studied the PBPK
model developed by Andersen et al. (10) for methylene chloride (DCM),
which includes a total of 23 distinct parameters relating to tissue weights,
blood fiow rates, partition coefflcients, and metabolic constants. Rather
than focusing directly on the metabolites of methylene chloride reaching
the lung and the liver, Portier and Kaplan (19) used the PBPK model
predictions of metabolite concentrations in these two tissues to estimate
the lifetime cancer risk due to exposure to methylene chloride. In this
analysis, an essentially linear pharmacodynamic model was used to estimate
cancer risks, with tissue doses expressed in terms of the area under the
concentration-time curve in lung or liver for metabolites of DCM produced
by the glutathione-S-transferase (GST) pathway.
Plausible ranges of uncertainty for the PBPK model parameters were based
on published data when available; otherwise, coefficients of variation of
the parameters were arbitrarily assigned values of 20 to 200%. Portier and
Kaplan (19) reported that the variability in the 10-6
RSD (the dose estimated to increase the lifetime cancer risk by one in a
million) was substantially greater allowing for uncertainty in the PBPK
model parameters in comparison with treating these parameters as known constants.
Specifically, the standard deviation of the distribution of RSDs was increased
by a factor of about 10-fold.
Farrar et al. (20) conducted a similar uncertainty analysis for
perchloroethylene using the PBPK model similar to that used in modeling
styrene by Ramsey et al. (21). Because certain model parameters were
not independent of one another, multivariate probability distributions were
used to characterize prior information on parameter uncertainty. This study
focused on three measures of tissue dose, namely the areas under the concentration-time
curves for TCE in the liver and arterial blood, and the area under the concentration-time
curve for metabolites of TCE in the liver. In addition, the variability
in the RSD based on the induction of hepatocellular carcinomas was also
considered. Although the results were generally supportive of the findings
of Portier and Kaplan (19), these investigators concluded that the
choice of an appropriate measure of tissue dose was of relatively greater
importance for cross species extrapolation than uncertainty in PBPK model
parameters.
Subsequent investigations have also provided useful information on uncertainty
associated with predictions of tissue doses based on PBPK models. Hattis
et al. (22) reported appreciable differences in predictions of the
metabolism of perchloroethylene based on PBPK models constructed by seven
different groups of investigators. Although there were some structural differences
in the models used, the most important factor appeared to be the data used
to calibrate metabolic parameters. Bois et al. (23) noted that three
independently developed PBPK models for benzene provided noticeably different
fits to the same data on benzene metabolism. It was further shown that acceptable
fits to this data could be obtained with a relatively wide range of parameter
values. Hetrick et al. (24) conducted a sensitivity analysis of PBPK
models developed for styrene, methylchloroform, and methylene chloride.
Predictions of tissue doses were shown to be particularly sensitive to the
maximum rate of (Michaelis-Menten) metabolism and blood-air and blood-fat
partition coefficients. The degree of sensitivity was shown to depend on
the dose of the parent compound, the time at which tissue doses were predicted,
and the species for which the PBPK model was developed.
Applications of PBPK Modeling
In the examples discussed below the resolution of many of the mechanisms
involved in the uptake, distribution, metabolism and persistence of chemicals
in the body have been adequately resolved by the application of physiologically
based models. Although some have been only partially resolved, the process
of building a PBPK model often raises various questions that need to be
addressed to elucidate pharmacokinetic mechanisms.
Methylene Chloride
The PBPK model for methylene chloride derives from a relatively simple
model for volatile compounds originally developed for styrene (10,21).
This perfusion-limited model incorporated five principal compartments: the
lung, both as a compartment of excretion and, in some applications, the
site of uptake; the liver, as the principal site of metabolism; richly perfused
tissues, such as the kidney, brain and heart; slowly perfused tissues, such
as muscle and skin; and fat, which could play an important role in storage
and redistribution. Partition coefficients for blood and tissues were determined
by in vitro techniques (25,26).
Metabolism of methylene chloride, measured by in vitro and in
vivo techniques, was shown to proceed by two distinct pathways (26,27).
One pathway involved the mixed-function oxidases (MFO) and the other was
mediated by cytosolic GST. The MFO former pathway was found to be saturable
in rats and mice at inhalation exposures of greater than about 200 ppm over
a 6-hr period. The GST pathway was not saturable at concentrations up to
10,000 ppm over the same exposure period (26).
The delivered dose of the glutathione conjugated to the target tissues
in rats and mice (liver and lung) and correlated better with carcinogenic
response than did either the exposure dose or the products of metabolism
by the mixed function oxidase pathway. In vivo and in vitro
data from exposed humans and human tissues were used to calculate various
estimates of cancer risk in humans. A comparison of the physiologically
based pharmacokinetic model risk assessment data for liver tumors in humans
showed that it was 168 times less than that calculated using the conventional
U.S. Environmental Protection Agency (U.S. EPA) methodology, while the risk
for lung tumors was 143 times greater using the U.S. EPA approach (28,29).
Ethylene Oxide
Ethylene oxide (EtO) is used in the sterilization of medical devices
and as a fungicide in agriculture. It has also been identified as a human
metabolite ethylene, a common air pollutant (30). In the evaluation
of ethylene oxide as a carcinogen (31,32), the active carcinogenic
species was considered to be ethylene oxide per se with the formation and
elimination of metabolites representing a detoxification process (29).
The distribution of EtO was considered to be uniform throughout the body
because of the similarity of tissue-blood partition coefficients in different
compartments. It was also considered to be metabolized, principally by hydrolysis,
to varying degrees in all compartments (33). Hydrolysis, without
the intervention of enzyme catalysts, was shown to be followed by some degree
of conjugation with glutathione (34,35). In addition, EtO has been
shown to alkylate DNA and other biologic macromolecules (including hemoglobin)
by direct interaction (36,37).
The physiologic model used to fit blood and tissue concentration data,
was similar to that used for methylene chloride and styrene, except that
the brain and testes were isolated from the richly perfused tissue compartment
since these have been identified as target organs for EtO in animal studies
(38,39). Rate constants for glutathione conjugation and DNA adduct
formation were estimated from studies reported in the literature (40,41).
Data obtained in rats following iv administration and inhalation exposure
allowed the simulation of ethylene oxide concentrations in target tissues
as well as and the concentration of DNA and hemoglobin adducts. Experimental
data following the iv administration of up to 100 mg/kg of EtO or after
exposure to inhalation exposures to 1200 ppm for 6 hr did not appear to
demonstrate biochemical thresholds; whole body elimination of ethylene oxide
appeared to follow first-order kinetic process. However, this analysis facilitated
the identification and characterization of the hydrolysis of EtO, glutathione
conjugation, exhalation, and DNA or hemoglobin binding. These latter processes
were affected by saturation of elimination pathways at high-exposure concentrations
where the whole body elimination continued to follow first-order kinetics
(33). Information on the metabolic capacity of human tissues for
the hydrolysis and glutathione conjugation of ethylene oxide would greatly
facilitate the extrapolation of animal cancer risk estimates to humans.
1,4-Dioxane
1,4-Dioxane has been used for decades as an industrial solvent. Aside
from inducing hepatic and renal effects it has also been shown to induce
liver tumors in rodents and to induce nasal carcinomas in rats (42-44).
Young et al. (45) showed that the metabolism of 1,4-dioxane was saturable
at high doses.
Two recent reports on the application of physiologically based pharmacokinetics
of 1,4-dioxane (8,46) incorporated the kinetic constants for the
formation of the principal metabolite (ß-hydroxy ethylacetic acid).
The model accommodated the administration of 1,4-dioxane by the iv, inhalation
and oral routes, and allowed the calculation of the surrogate delivered
doses to various organs, particularly the liver. Metabolic thresholds were
observed at water concentrations of greater than 1% administered by the
oral route and at atmospheric concentrations of greater than 300 ppm inhaled
continuously (8). These investigators suggested that unless the physiologic
and metabolic differences between humans and rats were corrected for, human
cancer risk based on traditional risk assessment methods applied to the
administered dose would be overestimated.
1-Nitropyrene
1-Nitropyrene, a nitrated polycyclic aromatic hydrocarbon, has been detected
in diesel exhaust emissions, coal combustion products, and photocopier toners
(47-49). 1-Nitropyrene is closely related to the class of compounds
known as polycyclic aromatic hydrocarbons, many of which are known carcinogens.
The systemic uptake, metabolism and excretion of 1-nitropyrene has been
shown to be very rapid with wide distribution to body tissues (50).
1-Nitropyrene is a potent bacterial and mammalian mutagen, and induces
lung tumors in mice and mammary tumors in rats following subcutaneous injection
(51-53). Tumors were also found at the injection site.
Medinsky et al. (54) developed a PBPK model for 1-nitropyrene
incorporating the principal organs to which 1-nitropyrene was distributed,
metabolized and bound (namely the upper respiratory tract, lung, liver,
and kidney). This model provided a good description of the clearance of
metabolites via the bile and feces as well as through urine. Partition coefficients
for the lung, liver and kidneys relative to blood were found to be close
to unity, indicating that consideration of blood flow alone is sufficient
to describe the clearance of 1-nitropyrene from these tissues. This model
appeared to provide a good description of the doses of 1-nitropyrene and
its metabolites to target tissues, as well as their temporal relationship
postexposure.
Polychlorinated Biphenyls, Dioxins, and Furans
These classes of chemicals represent a major group of ubiquitous and
persistent environmental pollutants that have been a cause of great concern
for the past several decades (55). While the mechanisms involved
in the toxicity of these groups of chemicals are still under investigation,
immune alterations and decreased specific antibody response (56,57)
have been noted for the chlorinated biphenyls and 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) (58,59). The polychlorinated biphenyls tend to act as hepatocarcinogens
(44,60) induces liver tumors in rats and is cocarcinogenic (61,62).
The toxicity of the dioxins and furans differs markedly among species
(63,64) as does their pharmacokinetics and disposition (65,66).
The chlorinated dibenzodioxins and dibenzofurans accumulate in the liver
and adipose tissue, although there are notable species differences in their
relative distribution to these tissues (67). This may be due in part
to binding at different sites, such as the Ah locus, in responsive
and nonresponsive different species (68-70).
The PBPK models proposed for polychlorinated biphenyls (12,71),
2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofurans
(65,66,72,73) are all rather similar. They generally involve five
or six compartments, consisting of the blood, slowly perfused tissues, and
richly perfused tissues with separate compartments for the principal target
tissues: fat and liver.
For hexachlorobiphenyl, the model was able to describe blood level data
adequately following the administration of a single iv dose in the rat (12).
Since this compound is excreted very slowly, blood concentrations were followed
for 43 days postdosing. Growth, particularly of the fat compartment, was
incorporated into the model. The rate of conversion to, and excretion of,
the glucuronide conjugate via the bile was taken into account. Enterohepatic
recycling was considered to be important. The authors expressed the opinion
that the variables in the model had been adequately assessed and that species
and route variations could be accommodated to describe the delivered dose
to specific target tissues.
For the polychlorinated dibenzodioxins and dibenzofurans, bile rather
than urine was considered to be the major route of excretion. The liver
is a major site of toxic action of these agents. Liver-blood partition coefficients
were very large, ranging between 30 and 130 for the various rodent and primate
species considered. Microsomal enzyme induction and binding to the Ah
receptor sites were accommodated in the PBPK model. Since the arylhydrocarbon
hydroxylase complex has been implicated in the carcinogenic mechanism(s)
for these compounds, it has been suggested that the PBPK model could be
useful for cancer risk assessment (7,65,72-74).
Other Considerations in Applications of PBPK Models
The development of a PBPK model for a particular compound may require
special consideration of factors other than those involved in the applications
described in the previous section. One of the benefits of building a PBPK
model is increased understanding of the pharmacokinetic processes involved
in the distribution and elimination of xenobiotics. The examples that follow
illustrate the need for enhanced PBPK modeling to accommodate the effects
of growth and aging, topical adsorption, pregnancy, and competitive multiple
metabolites.
Animal Aging and Body Growth
In the studies of the pharmacokinetics of polychlorinated biphenyls discussed
earlier (12,71), it was found that hexachlorobiphenyl is excreted
very slowly by the rat. At 6 wk, postdosing, over 80% of the iv dose was
retained, and the fraction that was excreted in the urine and feces comprised
mainly of metabolites. Over this time period the rats, originally weighing
between 250 and 300 g, gained appreciable body weight. Since fat increases
proportional to total body weight when the animal grows, hexachlorobiphyl
in poorly perfused fat tissue became increasingly diluted during the experiment.
Thus, fat concentrations beyond 8 days postdosing were not well predicted
without the incorporation of an adjustment for the increase in the volume
of the fat compartment as a function of age.
A similar circumstance arose in attempts to fit data obtained from pharmacokinetic
and tissue disposition studies of methyl chloroform in young and old rodents
(75,76). To fit the data obtained from old (18.5 months) rats, it
was necessary to increase the size of the fat compartment from 7% (for young,
1- to 3- month-old rats) to 18% of their body weight. The concentration
of methylchloroform in body fat was 20 to 100 times greater than that in
any other tissue. Thus the model predicted that the increased size of the
fat compartment in older animals would increase the amount taken up. The
model also predicted that methyl chloroform would be more slowly released
in younger animals than in older animals. The same integrated model was
found to be applicable to data derived from iv administration, inhalation,
bolus gavage, and administration in drinking water, as well as data obtained
with repeated doses (8).
Topical Adsorption in Inhalation Studies
Inhalation studies of volatile chemicals have usually carried out by
placing the whole animals in a carefully controlled exposure chamber. In
the closed chamber technique (77,78) inferences about the kinetics
of uptake and disposition of volatile chemicals are made by monitoring the
change in chamber concentration with time.
In studying the kinetics of chlorinated ethane in rats, Gargas and Anderson
(79), encountered a high loss (78%/hr) of ethanes containing 3 or
more chlorine atoms from empty chambers. This turned out to be primarily
due to adsorption to carbon dioxide adsorbents and to the inner chamber
surface. While these effects could be measured and satisfactorily incorporated
into the PBPK model, the concentration of 1,1,2,2-tetrachloroethane, in
the chamber immediately following exposure, was underesti- mated by nearly
an order of magnitude.
Since the excess chamber concentration could be accounted for only as
an additional amount carried to the chamber passively by the animal, it
was assumed that tetrachloroethane was adsorbed extensively to the fur of
the animals. This hypothesis was confirmed by exposing rats to 350 ppm of
tetrachloroethane for 6 hr in an exposure chamber. The animals were then
given a lethal dose of sodium pentobarbital and placed singly in an exhaled
breath chamber with the chamber atmosphere monitored for tetrachloroethane.
Since the animals were not breathing, it was assumed that the atmospheric
concentrations of tetrachloroethane could only have been derived from the
desorption of the chemical from the fur of the animal. This experiment allowed
the derivation of the amount of the chemical adsorbed on the fur and a first-order
rate coefficient was determined for desorption from the fur. When the fur
adsorption/desorption data was used in conjunction with exhaled breath analysis,
the PBPK provided an adequate fit of the live animal data.
Pregnancy, Lactation, and Nursing
Many changes occur during pregnancy that can have a significant impact
on the toxicodynamics of a particular chemical. For example, the changes
in body weight, total body water, plasma proteins, body fat, and cardiac
output will alter the distribution of many xenobiotics (80-82).
PBPK models have been used to describe the kinetics and disposition of
the drugs tetracyline, morphine, and methadone (83-85). Two recent
reports described the pharmacokinetics and disposition of trichloroethylene
and its principal metabolite, trichloroacetic acid, in the pregnant rat
as well as the lactating rat and nursing pup. A PBPK model consisting of
eight compartments for trichloroethylene in the pregnant rat and nine compartments
for trichloroacetic acid was used (86). Both models accommodated
multiple routes of exposure and repeated dosing. The model provided for
a variable litter size 1 to 12 pups per rat as well as placental growth.
Values of the maximum rate of metabolic removal velocity (Vmax)
in naive and pregnant rats were 10.98 and 9.18 mg/kg/hr, respectively. This
reduction is significant, and has been related to a decrease in the cytochrome
P450 monooxygenase activity due to altered steroid hormones. A high substrate
affinity was demonstrated. Although the value of the Michaelis constant
(Km) was low (0.25 mg/l), it was similar in both groups
of rats. Fetal exposure to trichloroethylene was estimated to range from
67 to 76% of the maternal exposure; fetal exposure to the trichloroacetic
acid metabolite was 63 to 64% of that of the dam.
The model-fitted data obtained after exposure by the inhalation, oral
gavage, or by drinking water. Other kinetic parameters predicted by the
model (such as the relative volumes of distribution, the peak blood concentration
following oral gavage, fetal concentrations following inhalation exposure,
absorption, and elimination rates) agreed well with previous data reported
in the literature. These results demonstrated that fetal exposure to both
the parent compound and its principal metabolite was significantly elevated
in relation to the maternal exposure.
Fisher et al. (87) examined the transfer of trichloroethylene
and trichloroacetic acid to nursing pups from lactating dams exposed to
trichloroethylene by inhalation of 610 ppm, 4 hr/day, 5 d/week from days
3 to 14 of lactation. A further study involved exposure of the lactating
dam to 333 mg/ml of trichloroethylene in drinking water from days 3 to 21
of lactation. The exposure of the pups to trichloroethylene was solely from
ingested maternal milk; however, their exposure to trichloroacetic acid
derived from maternal milk and from metabolism of ingested trichloroethylene.
The model provided for different values during lactation for compartmental
volumes, blood flows, and milk yield obtained from the published literature.
Metabolic and other kinetic parameters were determined experimentally.
The values of Vmax = 9.26 mg/kg/hr in the lactating
rat was similar to that in the pregnant rat. However, the value of Vmax=
12.94 mg/kg/hr obtained for male and female pups indicated that the ability
of the pups to metabolize trichloroethylene was greater than that of the
adult. The plasma half-life (16.5 hr) of trichloroacetic acid in the pups
was also substantially greater than that of the mature rat. Unlike most
other physiologic distribution processes which are flow limited, the distribution
of trichloroacetic acid to mammary tissue is diffusion limited. The exposure
of the pups to trichloroethylene from maternal milk was small, representing
only about 2% of the exposure of the dam. Pup plasma levels of trichloroacetic
acid; however, were as high as 30 and 15% of the maternal exposure for drinking
water and inhalation exposures, respectively.
Mixtures
The interaction between two or more chemicals is a very complex phenomenon
involving chemical interactions prior to absorption, competition for a common
metabolic pathway, and competition for sites of toxic action (88).
Each of these processes could be a consequence of other complex mechanisms
that have seldom been defined completely.
One example, in which PBPK modeling has been used to describe the quantitative
interaction between two compounds metabolized by the same microsomal oxidation
pathway is that of trichloroethylene and 1,1-dichloroethylene (89).
1,1-Dichloroethylene was shown to induce acute hepatoxicity as a consequence
the reactivity of their metabolic products. The metabolic pathway was the
same for both compounds, and there was high-affinity substrate binding which
was saturable (90). Coexposure of rats to 1,1-dichloroethylene and
vinyl chloride dramatically reduced the hepatoxicity of the former (91).
A PBPK model was developed, assuming competitive inhibition by both compounds,
to estimate the hepatoxicity of 1,1-dichloroethylene in the absence and
presence of trichloroethylene. Elevated liver enzymes provided a pharmacodynamic
index of hepatotoxicity. In rats exposed to 0, 200, 300, and 400 ppm of
dichloroethylene alone, it was observed that the serum glutamic oxaloacetic
transaminase (SGOT) enzyme level increased dramatically at exposures above
100 ppm. When animals were exposed to 300, 713, and 1718 ppm of 1,1-dichloroethylene
and 500 ppm of trichloroethylene, the SGOT elevation was significantly reduced.
This behavior was consistent with purely competitive inhibition with binding
constants of 0.25 mg/l for trichloroethylene and 0.10 mg/l for 1,1-dichloroethylene.
It was evident that 1,1-dichloroethylene is a slightly better substrate
for microsomal oxidation than trichloroethylene. The model was able to predict
the combined pharmacodynamic effects of these two substrates when coadministered
in any proportion.
Pharmacodynamics
Chemical carcinogenesis is a complex process involving a number of steps,
which may include biotransformation of the parent compound to its reactive
metabolites, DNA damage, mutation, proliferation of mutated cells, progression
to a malignant state, and growth of malignant tissue to overt tumors (66,92).
The two-stage clonal expansion model of carcinogenesis, originally developed
by Moolgavkar and Venzon (93) and Moolgavkar and Knudson (94)
provides a convenient framework for describing the process of carcinogenesis.
The model is based on the premise that two critical mutations are required
to convert a normal stem cell to a malignant cancer cell; the effects of
cell kinetics are reflected in the birth and death rates of stem cells as
well as initiated cells which have sustained the first mutation.
The dose of the proximate carcinogen to the target tissue can impact
the process of neoplastic transformation in several ways. The probability
of either a first- or second-stage mutation can be increased by direct alkylation
of DNA at the time of cell division. Mitogenic compounds which increase
the rate at which stem cells divide can also increase the mutation frequency
by increasing the opportunity for either spontaneous or induced mutation.
Once a genetic lesion induced in a stem cell has been fixed by replication,
promoting agents may augment the size of the iniated cell population by
selective clonal expansion of initiated cells. Increasing the pool of initiated
cells increases the number of cells at risk of sustaining the second critical
mutation, which completes the process of neoplastic conversion. This two-stage
model of carcinogenesis is no doubt an oversimplification of the process
of neoplastic transformation. Nonetheless, it does embody critical factors
such as mutation, tissue growth, and cell kinetics involved in carcinogenesis,
and has proven useful in modeling dose-response relationships observed in
toxicologic and epidemiologic studies (1).
An important consideration in such applications is the manner in which
dose in incorporated into the modeling process. In most applications, the
external level of exposure to the host has been used in predicting cancer
risk. The use of the external level of exposure in dose-response modeling
can lead to biases in predictions of low-dose cancer risks when the relationship
between the administered dose level and the dose of the reactive metabolite
reaching the target tissue is nonlinear (95). Physiologic models
offer an approach to tissue dosimetry which can be used to avoid such biases.
PBPK models permit calculation of various measures of tissue dose. With
a given chemical carcinogen, the mechanisms of interaction between dose
and tissue constituents and the mechanisms by which these interactions lead
to cancer will determine the proper measure of tissue dose for risk assessment
calculations. Within the framework of the two-stage model, increases in
either mutation rates or growth rates of normal or intermediate cells can
lead to increased cancer risk. It follows that the interaction of the toxicant
with target tissue must have cellular consequences that are eventually reflected
in alterations in the rates of one or more of these processes.
Mechanistic information has been used to suggest a very broad brush classification
of chemical carcinogens (92). Genotoxic carcinogens interact directly
with DNA bases to form promutagenic adducts which alter mutational probability
during cell division. Nongenotoxic carcinogens do not interact directly
with DNA; rather, they alter cell division rates. These alterations can
either be due to direct mitogenic stimulation of the tissue or indirectly
as a reparative process following tissue cytotoxicity. Because all carcinogens
interact with cells leading to formation of genotypically altered cells
and must therefor have some genotoxic sequelae, the terms DNA reactive and
non-DNA reactive have also been suggested as a more appropriate than genotoxic/nongenotoxic.
Another proposal is the differentiation between genotoxic and operationally
nongenotoxic carcinogens. In general DNA reactive carcinogens are initiators
and the non-DNA reactive chemicals are promoters. The mechanism of toxicant-tissue
interaction specifies the linkage between dosimetry of these individual
carcinogens and the rates of mutation or of proliferation of normal and
altered cells involved in tumor formation. The linkage between tissue dose
and these cellular parameters for three broad classes of chemical carcinogens
is examined below.
DNA Reactive Chemicals
Certain electrophiles react with nucleophilic sites on DNA bases to form
promutagenic adducts. Chemicals such as ethylene oxide, propylene oxide,
dimethylmethylsulfonate, and bis-chloronitrosourea react directly with these
bases (96,97). Other chemicals such as nitrosamines, vinyl chloride,
and aflatoxins have to be metabolized in the body to form reactive electrophiles
(96). In either case, tumor formation is believed to be associated
with changes in mutation probabilities related to the production and persistence
of adducts. The adducts are generally expected to be formed by second-order
reactions and removed by saturable repair enzymes. For a chemical like ethylene
oxide (EtO), the equation for the rate of change in the amount of tissue
adducts over time t is (33):
[22]
Here, [EtO], [DNA], and [DNA-EtO] denotes the concentration of EtO, DNA,
and DNA-EtO adducts in the target tissue with volume V. The parameter
k represents the second-order rate constant at which adducts are formed,
with Vmax and Km denoting the Michaelis-Menten
parameters for (saturable) enzymatic repair.
With this model, [DNA-EtO] can be used as a surrogate for increases in
probability of mutation in each cell division event. Highly reactive chemicals
will form several different adducts and the mutational efficacy will vary
for each of these individual adducts. However, if all processes are linear,
the increased mutational rates would still be expected to be linearly related
to the integrated tissue exposure to EtO. In this case, the cumulative exposure
of the target tissue to the critical reactive metabolite may be used as
a measure of tissue dose. Other factors need to be considered in interspecies
extrapolation. For example, it is unclear whether risk across species for
DNA reactive chemicals should be normalized based on integrated daily dose
or on integrated lifetime dose. In either case, the tissue dose metameter
is the integrated tissue dose of DNA reactive electrophile integrated over
an appropriate period of the animal or human life span.
Non-DNA Reactive Cytoxic Chemicals
With non-DNA reactive chemicals, the initial cellular interactions are
frequently associated with accumulation of sufficient doses of highly reactive
species to cause cell death. The parent compound may be reactive, as with
formaldehyde in the nasal mucosa or ethyl acrylate and ethylene dichloride
in forestomach (97,98). In other cases, the chemical is converted
to reactive metabolites in the target tissues: chloroform, carbon tetrachloride,
and vinylidene chloride kill hepatocytes only after metabolism to phosgene,
trichloromethyl free radical, and chloroacetylchloride, respectively (99-101).
The mechanisms of toxicant-target tissue interaction vary depending on the
chemical nature of the reactive intermediate. Some reactive chemicals form
long-lived stable adducts with cellular proteins and lipids. Examples include
carbon tetrachloride and acetaminophen (99,102). The depletion of
critical tissue macromolecules below critical levels then leads to cell
death. For carbon tetrachloride (CCl4), a reactive trichloromethyl
free radical (CCl3) covalently binds to macromolecules. The two
equations relating tissue-macromolecule [tissue-MM] concentrations include
terms for MM synthesis (k0), MM degradation (k1),
loss of MM due to a second-order reaction with reactive chemical (k2),
accumulation of the tissue-CCl3 adducts from the second-order
reaction, and degradation of the adducts (k3):
[23]
and
[24]
The concentration of free radicals should be related to the reaction
rate divided by the tissue volume (10). In this example, biologic
factors such as the MM synthesis rate and degradation rate constant of adducted
protein and lipid are important factors in interspecies extrapolation. Cell
death is expected to be associated with reduction of tissue MM below critical
levels for some period of time. A PBPK model of CCl4 has been
used to estimate tissue burdens of bound reactive metabolite in rats (103),
although it has not yet been applied in a risk assessment calculation. Cis-platin,
a cancer chemotherapeutic agent, forms protein adducts which have a cell
half-life similar to the cell half-life estimated for the CCl3
adducts from carbon tetrachloride. Farris et al. (104) have developed
a detailed PBPK model of cis-platin that describes adducts with both
low molecular weight and high molecular weight peptide constituents in cells.
Unlike CHCl3 which does not react appreciably with DNA, cis-platin
cytotoxicity is believed to be related causally to its cross-linking with
DNA that interferes with cell replication.
With CHCl3, a PBPK model has been developed to estimate cell
killing under various exposure conditions in rats and mice (105).
CHCl3 is metabolized to short-lived intermediates, phosgene and
hydrochloric acid, which are highly irritant, but whose adducts are not
persistent (106).
Cell death was more closely associated with high rates of metabolism
over relatively short times then to persistence of macromolecular adducts
(105,107). An empirical approach was taken to describe the relationship
between the rate of death of normal hepatocytes (-dNh/dt)
and the rate of metabolism per unit volume of tissue (dAmet/dt)/V).
The rate of loss of hepatocytes was modeled with a rate constant for cell
death (Kdeath), the number of viable hepatocytes at any
time (Nh), and a distribution of sensitivities to cell killing (SENS)
that was based on the rate of metabolism per unit tissue volume:
[25]
The sensitivity distribution described the proportion of cells at risk
at any rate of chloroform metabolism in the cell and was derived empirically
from appropriate experiments. In the absence of chronic tissue injury, cell
replication should restore the tissue to normal functional status. The cell
replication rate should mirror the death rate with some delay. The replication
rate affects the overall mutation rate, which corresponds to the product
of mutation probability per cell division and the rate of cell division.
Many chemicals will resist such simple classification, and demonstrate
characteristics shared by multiple categories. Nitrosamines are highly mutagenic,
but are also cytolethal at high doses. These chemicals alter both mutational
and replicative processes in cells in the target tissues (108). The
mutational efficacy persists even at doses below which there is little cell
killing. Formaldehyde is cytolethal and produces DNA-formaldehyde-protein
cross-links which probably have mutagenic potential (109). The exposure
response relationships for both processes are nonlinear with similar shapes.
The mutation rate for chemicals with cytotoxic and DNA reactive characteristics
should be related to the product of replication rate times the level of
cross-links; the increases in replication rate should affect other cell
growth parameters as well (110).
Non-DNA Reactive Mitogenic Chemicals
It is difficult to define the action of this diverse class of carcinogens
by a single mechanism. These compounds appear to promote tumor development
primarily by increasing cell replication in the two-stage cancer model,
and frequently by selective enhancement of replication in preneoplastic
instead of normal tissue. Many of these chemicals act via receptor molecules
to modulate expression of protein growth factors. Examples include dioxin,
dioxinlike polyhalogenated biphenyls and dibenzofurans, peroxisomal proliferators,
phenobarbital, and phenobarbitallike PCBs, as well as various hormones and
hormone-analogs. The relevant measure of biologic dose leading to cell replication
is the alteration in concentration of protein-growth regulatory products.
The relevant measure of tissue dose of chemical must be related to interactions
between the chemical and the receptor molecules responsible for regulating
expression of these growth factors.
PBPK models for dioxin have attempted to link dioxin tissue concentrations,
receptor action, and expression of particular genes. To date, the models
account for expression of certain metabolizing proteins, cytochrome P4501A1
(CYP1A1) and cytochrome P4501A2 (CYP1A2), but not for induction of specific
sets of growth factors. Dioxin interacts with a cytosolic protein, the Ah
receptor, and the dioxin-receptor complex translocates to the nucleus
and, together with at least one other protein factor, binds to specific
sites on DNA to modulate transcription of various genes (59,111).
Several receptor-based models linking tissue dioxin and gene regulation
have been described. Initially, induction was calculated by estimating the
fractional occupancy of the Ah receptor, as determined based on an
Ah receptor-dioxin binding constant, and the tissue concentration
of dioxin and Ah receptor (46,112). More recently, this induction
model has been extended in an attempt to account for ternary interactions
involving the binding of the dioxin-Ah receptor complex to sites
on DNA and the possibility of cooperative interactions among multiple DNA
binding sites for this complex in regulatory regions of specific genes (112).
The relationships modeled include the binding of dioxin and Ah receptor,
cooperative binding of the receptor-dioxin complex to DNA sites, and the
increased synthesis rate of protein consequent to the alterations in gene
transcription. The two relevant relationships become
and
Here, Kb is the dioxin-Ah receptor dissociation
constant, K0 is the basal synthesis rate of CYP1A1, Kmax
is the fully induced CYP1A1 synthesis rate, n is a Hill coefficient
interpreted as reflecting the cooperative binding of the Ah receptor-dioxin
complex to regulatory regions of DNA, Kd is the dissociation
constant for the DNA-Dioxin-Ah complex, and K1
is the degradation rate constant for the CYP1A1 under normal conditions.
To be of use in risk assessment, these models will ultimately have to predict
the regulation of sets of growth regulatory genes in an attempt to model
the proliferative responses directly. In the interim, the induction of specific
gene products, such as CYP1A1 and CYP1A2, can be used to develop correlations
with carcinogenic sequelae of these promoters, bearing in mind that there
is no reason to expect a causal relationship between these cytochrome activities
and tumor formation.
Summary and Conclusions
In this article, we have reviewed the development and application of
PBPK models as a tool for estimating the dose of reactive metabolites of
chemical carcinogens reaching target tissues. In general terms, PBPK models
envisage the body as being comprised as a small number of relatively homogeneous
physiologic compartments. Such models are characterized mathematically by
a system of mass-balance equations that can be readily solved using modern
computer software to obtain predictions of tissues doses. However, the development
and applications of a PBPK model is not a trivial undertaking. Information
on all of the allometric, biochemical, and pharmacokinetic parameters involved
in the model must be developed, and the model validated and refined by appropriate
experimentation. This process can generate significant insight into the
uptake, distribution, metabolism, and elimination of xenobiotics suspected
of increasing cancer risk.
From the risk assessment point of view, the primary goal of PBPK modeling
is to obtain more accurate estimates of cancer risk through the use of more
accurate measures of tissue dose. While the use of more relevant measures
of dose is likely lead to progress towards this objective, the uncertainty
associated with predictions of tissue dose must not be overlooked. This
uncertainty can be evaluated by considering the precision associated with
each of the model parameters, and by identifying those parameters to which
predictions of tissue dose are most sensitive.
Complete, quantitative biologically motivated models for carcinogenic
risk assessment must ultimately include several components: a PBPK description
for tissue dosimetry, a linking model specifying the mechanism by which
tissue dose interacts with cell constituents to produce alterations in cell
growth rates or mutation probabilities during replication, and the impact
of alterations in these cellular events on tumor promotion. Significant
progress is evident in development of PBPK models and in investigating model
sensitivity and the impact of parameter variability on risk calculations
(19,23,113,114). The two-stage model is a promising quantitative
description of the relationship between cellular events and cancer which
can be expanded as new information on the obligate mutational events in
chemical carcinogenesis for specific carcinogens becomes known in more detail
(93,115,116). The greatest challenge today is the further elaboration
of these linkage processes that are important in transducing chemical interactions
into direct biologic consequences. Perhaps the most tangible reward expected
from improved quantitative linking models is the more precise definition
of the measure of tissue dose and the possibility of better definition of
the manner in which these measures of tissue dose should be normalized across
species for various mechanisms of action to support informed interspecies
extrapolations.
The work required to develop these comprehensive, biologically motivated
risk assessment models including their validation with specific experiments
under long-term chronic exposure conditions is expected to be costly and
time-consuming. Clearly, it is impossible to create these models for every
chemical. A more reasonable, cost-effective strategy may be to invest in
models for a limited number of chemicals with well-defined mechanisms of
action in order to create prototype risk assessment approaches for generic
classes of chemicals of widespread interest. With these prototype chemicals,
model development early on in the process of toxicity testing can guide
subsequent experimental design for validating, refuting, or refining current
modeling methodologies. These linking models are now in their formative
stages, and more work will be required to improve the biologic basis of
these descriptions and test various assumptions. Their very existence, though,
will help refine and focus succeeding research efforts and improve the likelihood
that research will find more fruitful the process of quantitative risk assessment
for chemical carcinogens.
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