Metabolism of Trichloroethylene
Lawrence H. Lash,1 Jeffrey W. Fisher,2 John C. Lipscomb,3 and Jean C. Parker4
'Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan USA; 2Toxicology Division, Armstrong
Laboratory, Wright-Patterson AFB, Ohio USA; 3National Center for Environmental Assessment, U.S. Environmental Protection Agency,
Cincinnati, Ohio USA; 4National Center for Environmental Assessment, U.S. Environmental Protection Agency, Washington, DC USA
A major focus in the study of metabolism and disposition of trichloroethylene (TCE) is to identify
metabolites that can be used reliably to assess flux through the various pathways of TCE
metabolism and to identify those metabolites that are causally associated with toxic responses.
Another important issue involves delineation of sex- and species-dependent differences in
biotransformation pathways. Defining these differences can play an important role in the utility of
laboratory animal data for understanding the pharmacokinetics and pharmacodynamics of TCE in
humans. Sex-, species-, and strain-dependent differences in absorption and distribution of TCE may
play some role in explaining differences in metabolism and susceptibility to toxicity from TCE
exposure. The majority of differences in susceptibility, however, are likely due to sex-, species-, and
strain-dependent differences in activities of the various enzymes that can metabolize TCE and its
subsequent metabolites. An additional factor that plays a role in human health risk assessment for
TCE is the high degree of variability in the activity of certain enzymes. TCE undergoes metabolism
by two major pathways, cytochrome P450 (P450)-dependent oxidation and conjugation with
glutathione (GSH). Key P450-derived metabolites of TCE that have been associated with specific
target organs, such as the liver and lungs, include chloral hydrate, trichloroacetate, and
dichloroacetate. Metabolites derived from the GSH conjugate of TCE, in contrast, have been
associated with the kidney as a target organ. Specifically, metabolism of the cysteine conjugate of
TCE by the cysteine conjugate ,B Iyase generates a reactive metabolite that is nephrotoxic and may
be nephrocarcinogenic. Although the P450 pathway is a higher activity and higher affinity pathway
than the GSH conjugation pathway, one should not automatically conclude that the latter pathway is
only important at very high doses. A synthesis of this information is then presented to assess how
experimental data, from either animals or from in vitro studies, can be extrapolated to humans for
risk assessment. Key words: conjugate beta-lyase, cysteine glutathione, cytochrome P450,
glutathione S-transferases, metabolism, sex dependence, species dependence, tissue dependence,
trichloroethylene. - Environ Health Perspect 1 08(suppi 2):177-200 (2000).
http.//ehpnetl.niehs. nih.gov/docs/2000/suppl-2/1 77-2001ash/abstract.html
An understanding of the importance of each
pathway of trichloroethylene (TCE) metabolism is critical for determining susceptibility,
target organ specificity, and extrapolation of
animal data to humans. Both a qualitative
and a quantitative understanding of sex and
species differences in metabolism should
allow more accurate physiologically based
pharmacokinetic (PBPK) modeling to be
done and, hence, more accurate risk assessments to be made. Of course, the ability to
obtain some metabolism data from human
tissue enables comparison of modeling data
with actual data and assessment of the accuracy of the PBPK models. The final section of
the review highlights some of the principal
remaining questions and research needs relating to metabolism of TCE.
A scheme summarizing the major metabolic pathways for TCE is shown (Figure 1).
As described below and in other reviews in this
monograph, the metabolic flux through the
oxidative and glutathione (GSH)-dependent
pathways differ in each tissue. Hence, modes
of action for each target organ, which are
largely dependent on the chemical species
generated, will differ. Most of the focus on
the oxidative pathway has been on the liver,
which has the highest activities of any tissue
of the various isoforms of cytochrome P450
(P450). P450-derived metabolites have been
directly associated with liver injury. The
lungs are additional target organs for which
P450-derived metabolites have been linked to
toxic and pathologic processes. Although
P450 activity is present in the kidneys, to
date the nephrotoxic and potential nephrocarcinogenic effects of TCE have only been
associated with metabolites derived from the
GSH conjugation pathway. As described in
subsequent sections of this review, the reactions of the GSH conjugation pathway, with
the exception of the initial conjugation step,
which occurs predominantly in the liver but
can also occur in extrahepatic tissues, occur in
the kidneys. This is caused by tissue-selective
localization of transport processes and bioactivation steps (i.e., the cysteine conjugate
3-lyase [,-lyase]) in epithelial cells of the
renal proximal tubules.
For the ultimate purpose of human
health risk assessment, it is important to put
the metabolism data in the context of actual,
potential exposures. Hence, for most
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
environmental exposures to TCE, such as
those that may occur with contaminated
drinking water, rates of metabolism in
humans may be extremely small because of
high Km and low V. values relative to those
found in rodents. At higher occupational
exposures to TCE, however, many of these
metabolic pathways may be quantitatively significant in humans as well. A discussion of
exposure levels in the human population is
beyond the scope of this review. Readers are
referred to the review by Wu et al. (1) on
TCE exposure assessment and the review on
PBPK modeling by Clewell et al. (2) in this
monograph for more detailed discussions of
exposures that are relevant to humans.
In Vivo Absorption and
Distribution of TCE
As a result of its chemical properties (e.g.,
volatility, lipophilicity), TCE is readily
absorbed across biological membranes. For
human exposures, TCE is considered to be an
eye and skin irritant. There are essentially
three types of exposures to consider for
humans or laboratory animals: inhalation,
dermal, and oral. Exposure is usually either
from TCE vapor or from TCE liquid. In
either form, TCE is rapidly and extensively
absorbed through the lungs or gastrointestinal
tract, respectively. Absorbed TCE is then subsequently distributed to different target
organs (e.g., lungs, liver, kidneys, nervous system) via the circulatory system.
After inhalation exposure, TCE is rapidly
and extensively absorbed through the alveolar
endothelium due to a high blood/gas partition coefficient. Although absorption is high
in all cases, blood/gas partition coefficients
vary significantly among species (Table 1).
Since the blood/gas partition coefficient in
humans is approximately 1.5- and 2.5-fold
lower than that in mice and rats, respectively,
This article is part of the monograph on Trichloroethylene
Toxicity.
Address correspondence to L.H. Lash, Dept. of
Pharmacology, Wayne State University School of
Medicine, 540 East Canfield Ave., Detroit, Ml 48201.
Telephone: (313) 577-0475. Fax: (313) 577-6739.
E-mail: l.h.lash@wayne.edu
The views expressed in this article are those of the
authors and do not necessarily reflect the views or
policies of the U.S. government agencies.
Received 20 October 1999; accepted 21 January
2000.
177
LASH ET AL.
this suggests that delivery of TCE to the
circulatory system for translocation to target
organs may be significantly less efficient in
humans than in rodents and is a factor that
may need to be taken into account when
using animal data in a risk assessment analysis
for TCE.
Dermal absorption from exposure to TCE
vapor is negligible, although direct skin contact with TCE liquid may lead to significant
absorption. Since a large proportion of the
TCE that is absorbed dermally in humans is
excreted unchanged through the lungs and
this is not the usual route of exposure to
TCE, dermal absorption is not considered to
be a major factor in the risk assessment analysis (3). These data have been reviewed by
Davidson and Beliles (4). It should be noted,
however, that a study of dermal absorption of
dilute, aqueous solutions of TCE and similar
organic solvents in hairless guinea pigs indicated that significant absorption occurs (5).
This type of exposure mimics that of the
human population to drinking water contaminated with TCE. Hence, dermal absorption
should be considered in risk assessment analyses
for this type of exposure.
Besides inhalation, oral absorption is a
major factor in exposure to TCE. Since TCE
is uncharged, nonpolar, and highly lipophilic,
gastrointestinal absorption is extensive and
occurs by passive diffusion. One would
expect, therefore, that oral absorption would
be a nonvarying element in the pharmacokinetics of TCE. However, differences in oral
absorption of TCE are evident when TCE is
administered either in water or corn oil. For
example, Withey et al. (6) demonstrated that
peak blood concentrations of TCE in rats
given an intragastric dose (18 mg/kg) in water
was nearly 15-fold higher than those in rats
given the same dose in the same volume of
corn oil (14.7 vs 1.0 pg/mL). Hence, the
authors concluded that gastrointestinal
absorption of TCE can be limited by the
absorption of the vehicle. The vehicle used in
exposures in animal studies, therefore, needs
to be taken into account when extrapolating
animal exposure data for human health risk
assessment.
As a consequence of differences in blood
flow and overall metabolic rate, species differences exist in the fraction of administered
dose of TCE that is available for conversion
cI
cl
H
GST/
CI
1
P-450
0
OH.CH2-C,
[TCE-Oxide
?-
SG
Cl
NH-(CH220H
/
or
7*
6 TCE-O-P-4 50 /
H>=<Cl
e.
('
HO
OH
C12CH- C,
H
H
Cla=
0
(D
_
OH
C13C-C s
\H
C13C-CH"
'OH
2b
j3-Lyase
I
C13C
HN~
Cl
S
H>=<Cl j
4
Ci
:0aC
CCC13CCH20H
\OH
1*I
C12CH -C\OH
.~~~~~~~~~~~
10Q *
Ci3CCH20-gluc
CICH
Cytotoxicity
Mutagenicity
4H
14*
HO
OH
Figure 1. Scheme of metabolism of TCE. Metabolites marked with an asterisk are known urinary metabolites.
Metabolites: 1 = TCE; 2 = DCVG; 3 = DCVC; 4 = 1,2-dichlorovinylthiol; 5 = NAcDCVC; 6 = TCE-P450 or TCE-oxide intermediate; 7 = N-(hydroxyacetyl)-aminoethanol; 8 = oxalic acid; 9a = chloral; 9b, chloral hydrate; 10 = dichloroacetic
acid; 11 = trichloroacetic acid; 12 = trichloroethanol; 13 = trichloroethanol glucuronide; 14, monochloroacetic acid.
178
to toxic metabolites in the target organs. For
example, Prout et al. (7) compared blood
concentrations of TCE, chloral hydrate
(CH), trichloroethanol (TCOH), and
trichloroacetate (TCA) over time after administration of a 1,000 mg/kg oral dose of TCE
to male Osborne-Mendel rats and male
B6C3F1 mice. They observed that blood concentrations for the three metabolites of TCE
were markedly higher in mice than in rats at
most time points, whereas those for TCE
were higher in rats than in mice, indicating
more rapid metabolism and elimination of
TCE in the mice (Table 2). Furthermore, the
area under the curve (AUC) was severalfold
higher for CH, TCOH, and TCA in mice
than in rats. Fisher et al. (8) also observed
approximately 5-fold higher peak blood concentrations of TCE in male and female rats
compared with male and female mice and
Table 1. Reported blood/gas coefficient for TCE in rats,
mice, and humans.
Species
Blood/gas coefficient Reference
Rat
25.8
F344, male
(163)
21.9
SD, male
(8)
21.9
F344, male
(7)
15.0
F344, female
(7)
Mouse
13.4
B6C3F1, male
(7)
14.3
B6C3F1, female
(7)
9.92
Human, both male
(163)
9.2
(164)
SD, Sprague-Dawley.
Table 2. Blood concentrations of TCE and selected
metabolites in male Osborne-Mendel rats and male
B6C3F1 mice after an oral dose of TCE.a
Peak blood
Time after
concentration
exposure
AUC
of peak (hr)
(pg/mL)
(mice:rats)
TCE
Rats
61.4
3.0
Mice
18.2
0.5
7.9
CH
Rats
0.86
9.0
Mice
1.77
3.0
1.0
TCOH
Rats
8.0
10.0
Mice
33.5
2.0
3.9
TCA
Rats
50
14.0
Mice 263
14.0
288
30.0
6.3
Rats and mice were given TCE (1,000 mg/kg) per os in corn oil.
Blood samples were taken by cardiac puncture and were analyzed by gas chromatography. AUC were determined graphically
up to the final time point taken (TCE: 7.5 hr in rats, 1 1 hr in mice;
CH: 15 hr in rats, 10 hr in mice; TCOH: 18 hr in rats, 8 hr in mice;
TCA: 40 hr in rats, 44 hr in mice). For TCA in mice, two major
peaks of blood concentration were observed. Data originally
reported in Prout et al. (7).
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
METABOLISM OF TCE
similarly higher peak plasma concentrations TCE and reported that 90-95% of the radioof TCA in male and female mice compared label was recovered in expired air and urine.
with male and female rats. These observations Dekant et al. (13) gavage-dosed mice and rats
have important implications for species differ- with 200 mg/kg of labeled TCE in corn oil
ences in susceptibility to toxicity. As discussed and recovered 93-98% of the radiolabel.
in the review on mode of action in the liver Prout et al. (7) administered several doses of
(9), TCA is believed to be the primary TCE labeled TCE (10-2,000 mg/kg) to rats and
metabolite responsible for liver injury and mice and recovered 93-98% of the radiolabel
proliferation. The markedly higher blood and reported that peak blood levels occurred
concentrations of TCA in mice compared in 1 hr in mice and 3 hr in rats. D'Souza et
with rats indicate that more TCA will get to al. (14), using classical pharmacokinetic
the target organ in mice and are consistent analysis, reported that oral and intravenous
with the male mouse as the sex and species bioavailability of TCE was 60-90% in nonthat is the most susceptible to liver injury and fasted rats and greater than 90% in fasted
carcinogenesis.
rats. Peak blood levels occurred between 6-10
In analyzing the distribution of TCE into min and blood concentrations were 2-3
various tissues, three major compartmental times higher in the fasted rats compared with
tissue groups can be identified: richly per- the nonfasted rats. Lee et al. (15) empirically
fused (e.g., liver, kidneys, lung), poorly per- demonstrated that presystemic elimination
fused (e.g., muscle), and adipose tissue. TCE (metabolism) of low dose rates of TCE was
readily equilibrates from the circulation into inversely related to dose and was nonlinear,
richly perfused tissues, with reported partition leading these authors to suggest that trace
coefficients for liver:blood or richly perfused amounts of TCE in the drinking water may
tissue:blood for male rats of approximately not enter the systemic circulation.
1.2 (8,10). Partition coefficients for female
No controlled oral ingestion studies in
rats (1.46) and for male and female mice human have been reported in the literature
(2.03 and 1.62, respectively) are modestly for TCE. Evidence for oral uptake of TCE in
higher than those in male rats (8), but these humans is inferred from case studies that
modest differences are not likely to be major report poisonings. Accidental or intentional
factors in the sex- and species-dependent dif- ingestion of large amounts of TCE has
ferences in TCE-induced toxicity. occurred, resulting in death (16). A more
Muscle:blood partition coefficients for TCE recent study of an attempted suicide of a 17have been reported to be less than 0.5 for year old who ingested 70 mL of TCE also
male and female rats and female mice and shows rapid systemic uptake and tissue distri1.00 for male mice (8,10). Hence, muscle is bution, with peak blood concentrations of
neither a major site of storage nor a major site TCE being obtained 13 hr after ingestion
of metabolism of TCE and does not signifi- (17).
Inhalation of TCE results in very rapid
cantly influence TCE pharmacokinetics or
uptake of TCE into the systemic circulation
pharmacodynamics.
Although the adipose tissue:blood parti- in animals (8) and humans (18), although
tion coefficient does not differ greatly among the time required to reach peak blood conmale and female rats and mice, with reported centrations differs across species. Peak blood
values ranging between 25 and 41 (8), the concentrations of TCE are achieved within
fact that it is so high indicates that sequestra- 1-2 hr in mice exposed to 100-750 ppmtion of TCE in fat may have a major influ- TCE vapors, in 4-6 hr in rats exposed to
ence on the pharmacokinetics of TCE. In 500-600 ppm TCE vapors, and in 8-12 hr
fact, the half-life of TCE in adipose tissue is in humans exposed to 100 ppm TCE.
Once TCE is in the systemic circulation,
estimated to be 3.5-5 hr, whereas that in
both the richly and poorly perfused compart- TCE is oxidized (metabolized) rapidly. Using
ments is estimated to be 2-4 min (4). Hence, PBPK models, estimates of metabolic capacslow release of TCE from fat several hours ity have been determined in rodents and
after initial exposures to TCE can lead to humans, and are summarized in Table 3. It is
continued delivery of TCE to target organs. clear that rodents have a much high capacity
Fat may also be a site of metabolism of TCE, to metabolize TCE than humans. Estimated
as cytochrome P4502E1 (CYP2E1) is present values for Michaelis-Menten affinity constants (Ki) were estimated to be low for
in adipose tissue microsomes (11).
rodents and humans (0.25-1.5 mg/L),
Pharmacokinetics of TCE
reflecting a high substrate (TCE) affinity.
and Its Major Oxidative
Although several metabolic products
Metabolites
derived from the GSH and P450 metabolic
As described above, orally administered TCE pathways have been identified for TCE, only
is rapidly and extensively absorbed into the a few of the major metabolites have been
systemic circulation. Daniel (12) dosed rats characterized pharmacokinetically in rodents
by stomach tube (60 mg/kg) with labeled and humans. The plasma half-life of TCA in
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
humans ranges from 86 to 99 hr after inhalation of either 50 or 100 ppm TCE, 6 hr/day
for either 5 or 10 days (19). Oral administration of either TCOH (10 mg/kg) or CH (15
mg/kg) in humans resulted in a 63- to 65-hr
plasma half-life for the metabolite, TCA.
Administration of TCA alone (3 mg/kg)
resulted in a plasma half-life for TCA of 51
hr (19).
The plasma half-life values for TCA are
much shorter in rodents than in humans. In
rats given intravenous administration of TCA
(5-6 mg/kg), the TCA plasma half-lives were
12 and 7 hr in males and females, respectively
(8). In mice given intraperitoneal doses of
TCA (5-10 mg/kg), the plasma half-lives for
TCA were 7 and 3 hr for males and females,
respectively (8). In mice exposed to TCE
vapors (42-889 ppm) for 4 hr, the estimated
plasma half-life values for TCA were 16 and 7
hr for males and females, respectively (8). In
male and female rats exposed to TCE vapors
(500-600 ppm), the estimated half-life value
for TCA in plasma was 15 hr (20).
The residence time for free TCOH in
blood is less than the residence time in
plasma for TCA in rodents and humans. The
half-life of free TCOH in blood is about 12
hr in humans exposed to 50 ppm TCE
vapors 6 hr per day for 5 days (21) and is 3
hr in mice administered 1,200 mg/kg TCE
via oral bolus intubation (22). Two other
oxidative end products readily found in blood
of mice administered TCE that are not readily measured in human blood are chloral
hydrate and trichloroethanol glucuronide.
CH and TCOH glucuronide (TCOG) were
not measured in humans exposed to 100 ppm
TCE for 4 hr (19). In mice gavage-dosed
with 1,200 mg/kg of TCE, the blood half-life
values for CH and TCOG were 3 and 5 hr,
respectively (22).
Oxidative Pathways of TCE
Metabolism
This section describes each step of the oxidative pathway of TCE metabolism and identifies key metabolites that are thought to be
important for development of toxicity or carcinogenesis. Although many of the enzymes
Table 3. Species-dependent differences in metabolic
capacity for TCE.a
Reference
Metabolic capacity (VmaxCJ
14.9
(15)
(16)
4-5
23.1
Female mice
(6,17)
32.7
Male mice
(6,17)
11-11.5
Male and
(6,17)
female rats
&Metabolic capacity ( VmaXC) is defined as the maximal rate of
Species
Humans
metabolism, with units of milligram substrate metabolized per
kilogram body weight per hour.
179
LASH ET AL.
that catalyze the specific steps of this pathway
are distributed fairly ubiquitously, a range of
activity levels of specific isozymes is found in
the different target tissues or in a given tissue
in males and females from various species,
which can markedly alter the distribution of
metabolites that are formed. This, in turn,
may have mechanistic implications as tissueand/or sex- and/or species-specific accumulation of certain metabolites can produce toxic
responses that are not otherwise observed.
liver microsomes. Furthermore, an epoxide
intermediate should yield CO, CO2,
dichloroacetate (DCA), and monochloroacetic
acid (CAA) as predominant metabolites from
TCE; in contrast to this, CH, TCA, and
TCOH are the major oxidative metabolites
that are recovered in both in vivo and in vitro
studies (28). Miller and Guengerich (27)
concluded that the species that irreversibly
binds to protein and DNA during TCE
metabolism must have, unlike an epoxide, a
reasonable degree ofchemical stability. Hence,
Descrption of Oxidative Pathways
the current dogma is that the majority of TCE
of TCE Metabolism
is believed to undergo chlorine migration in
Role offormation of TCE-epoxide in oxida- an oxygenated TCE-P450 transition state
tive metabolism of TCE. Although TCE is a leading to CH formation. However, Forkert
chemically simple compound, its metabolism and co-workers (29-32) have provided subis rather complex. The initial step in TCE stantial evidence that 1,1 -dichloroethylene,
metabolism, as described above (Figure 1), is which is very similar to TCE, is metabolized
either conjugation with GSH (discussed by P450 to an epoxide intermediate. These
below) or oxidation by P450. The oxidative findings suggest that the role of the epoxide in
pathway is quantitatively by far the major TCE metabolism should be revisited (see dispathway for TCE metabolism and the liver is cussion under "Questions and Research
the primary tissue where this step takes place, Needs" below).
The previous conclusion in the last U.S.
although P450 isoforms are present in most
tissues in varying forms and amounts. The Environmental Protection Agency risk assessinitial step in the oxidative metabolism of ment for TCE (33) that an epoxide intermemany halogenated alkenes is epoxidation diate is involved in the initial step of the
(23). Hence, the biotransformation of TCE oxidative metabolism of TCE may be erroto a reactive epoxide intermediate (2,2,3- neous but requires further study. In spite of
trichlorooxirane) was proposed many years this, a recent review on TCE metabolism and
ago (24,25). The finding of CH as a metabo- toxicity (34) showed TCE being converted
lite of TCE was seen as being consistent with by P450 to CH through an epoxide intermethe transient formation of a reactive epoxide diate and did not consider the oxygenated
intermediate. Epoxides can readily form acyl transition state and chlorine migration.
chlorides, which are subsequently hydrolyzed Uncertainty about the significance of an
to the corresponding acids, or they can form epoxide intermediate still exists, therefore,
aldehydes that are subsequently oxidized to and needs to be unambiguously resolved.
Metabolism of TCE to chloral hydrate
carboxylic acids or reduced to alcohols. The
existence of CH, TCOH, and TCA as major and trichloroethanol Four different P450 isometabolites of TCE is in accordance with this forms have been identified as playing a role in
scenario. Several years ago, controversy TCE metabolism: CYPlAl /2, CYP2B 1/2,
existed over whether TCE oxidation goes CYP2C1 1/6, and CYP2E1 (35-42). Of these
through an epoxide intermediate. The signifi- isoforms, CYP2E1 appears to be the major
cance of this is in the identification of the form with the highest affinity for TCE
electrophilic intermediate(s) that can cause (35,36,39), although considerable variability
cellular injury or mutations. An understand- can exist in the relative roles of different isoing of the chemical bioactivation mechanism forms, depending on physiological state and
is necessary for elucidating the mode of on the presence of other drugs or inducing
action of TCE in producing acute cellular agents. Furthermore, as discussed below, most
of the work on oxidative metabolism of TCE
toxicity and carcinogenesis.
In spite of the evidence in favor of epoxide has been done in the liver. Consequently, it is
formation, Miller and Guengerich (26,27) not known if isoform specificity for TCE is
concluded that TCE epoxide is not an oblig- the same in other tissues. Additionally, P450
ate intermediate in the formation of CH and isoform specificity in different animal species
that TCE epoxide cannot be the intermediate has not been thoroughly investigated.
responsible for irreversible binding to protein Differences among animal species in isoform
and DNA. Although the P450 heme is content and specificity may, and likely does,
destroyed during the oxidative metabolism of play a role in the observed differences in
TCE, the epoxide does not destroy the heme metabolism and toxicity.
(27). Green and Prout (28) also concluded
Lipscomb et al. (11) examined the oxidathat there was little evidence to support the tive metabolism of TCE in mouse, rat, and
intermediate formation of an epoxide in the human liver microsomes and showed that
oxidative metabolism of TCE in rat or mouse TCE alters the metabolism of several chemicals
180
that are characteristic substrates of CYP2E1 in
all three species (N-dimethylnitrosamine-Ndemethylase and p-nitrophenol hydroxylase),
of CYP3A (benzoxyresorufin Odeethylase) in
mice and rats, of CYPIA1/2 (ethoxyresorufin
Odeethylase and phenacetin O-deethylase) in
all three species, but had no effect on CYP2A
(coumarin hydroxylase) activity. The effect of
TCE was inhibitory for CYP2E1 and CYP3A
and stimulatory for CYPIA1/2. These data
confirmed earlier studies by Nakajima et al.
(40) that CYP2E1 is the major hepatic
isoform of P450 involved in TCE oxidation.
The broad range of halogenated hydrocarbons and other small organic molecules
that undergo oxidation by CYP2E1 and the
existence of several drugs and physiological or
pathological conditions that may lead to
induction of CYP2E1 indicate that certain
conditions or prior or concurrent exposure to
other chemicals, in particular ethanol, are
important factors that must be taken into
account in a risk assessment, since these may
markedly alter the capacity for TCE metabolism by this pathway.
After formation of the TCE-oxygen-P450
(TCE-O-P450) or epoxide intermediate, CH
(in equilibrium with chloral) is the major
metabolite produced. In the liver, only small
amounts of CH can be recovered as it is
rapidly converted to other compounds.
Hence, CH is not likely to be a major consideration for hepatotoxicity or hepatocarcinogenicity. In the lungs of male CD-1 mice,
however, TCE produces Clara cell injury
(43,44), and this has been attributed to the
accumulation of CH, which occurs because
the subsequent steps in the metabolism of
CH are much slower than the conversion of
TCE to CH (45). Besides having a slow rate
of subsequent metabolism of CH, the rate of
formation of CH in mouse lung is markedly
higher than that in either rat or human lung
(44). CH may be mutagenic, causing DNA
damage, and is clastrogenic, producing aneuploidy. Although CH has been shown to be
genotoxic (45), lung tumors resulting from
CH are primarily benign, which is more consistent with a nongenotoxic, cell-proliferative
mechanism. The cytotoxicity that can occur
from the accumulation of CH, with subsequent repair mechanisms, may enhance cell
proliferation leading to tumor formation. It
should be noted, however, that the cytotoxicity of CH requires high concentrations to be
accumulated, suggesting that the mouse lung
is a specialized case and that this mode of
action is probably not very relevant to that of
TCE in other tissues or to other species.
In addition to CH, oxalic acid (OX), N(hydroxyacetyl)aminoethanol, and DCA
(through formation of an acyl chloride intermediate) may be formed from the TCE-OP450 or TCE-epoxide intermediate (Figure
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
METABOLISM OF TCE
1). All four metabolites have been recovered
in the urine of both laboratory animals and
humans exposed to TCE. Subsequent metabolism is more complicated, involving multiple
steps and other oxidative and reductive
enzymes besides P450. With the exception of
DCA, which is also formed by other mechanisms (see below), the other two metabolites
are not likely to be of interest for understanding modes of action or risk assessment. CH is
further metabolized to either TCOH or TCA,
both of which can be further oxidized to
DCA. A pharmacokinetic analysis of CH
metabolism in B6C3F1 mice provided evidence that was consistent with some formation of DCA directly from TCA (46).
TCOH also undergoes glucuronidation, and
TCOG has been recovered in the urine of animals and humans exposed to TCE. TCOG
may also undergo enterohepatic recirculation
and regenerate TCOH (see below).
CH reduction to TCOH has been
reported to be inhibited by ethanol, and it
was suggested that this reaction is, therefore,
catalyzed by alcohol dehydrogenase (47,48).
A recent study in mouse liver microsomes
(49), however, found that pretreatment of
mice with pyrazole, which induces CYP2E1,
enhanced lipid peroxidation due to CH,
whereas addition of a general P450 inhibitor
reduced CH-induced lipid peroxidation. This
suggested that metabolism of CH to TCOH
and TCA is catalyzed primarily by CYP2E1.
Furthermore, a human lymphoblastoid cell
line expressing CYP2E1 metabolized CH to
mutagenic metabolites, proposed to be
derived from TCA and TCOH, whereas the
parent cell line, which lacks CYP2E1, was
inactive in CH metabolism (50). The proposed pathway of formation of the mutagenic
metabolites in the transfected human
lymphoblastoid cell line appeared to be similar to that in mouse liver microsomes. Since
ethanol is metabolized in liver by both alcohol dehydrogenase and CYP2E1 and Larson
and Bull (47) showed a dependence of the
reaction on NAD+, it is likely that both
enzymes can be involved. The precise role of
each enzyme in the reduction of CH to
TCOH, however, remains to be determined.
Schultz and Weiner (50) found that the
formation of TCOH from CH is stimulated
in vitro by inclusion of ethanol in the reaction
mixture. This, coupled with the in vivo data
of Sellers et al. (51), supports the role of alcohol dehydrogenase in the formation of
TCOH. The redox shuttling of cofactorenzyme complex in the sequential reduction
of CH and the oxidation of ethanol is
enabled by the rate-limiting release of cofactor. The apparent contradiction with studies
that have shown inhibition of alcohol dehydrogenase by CH may be explained by the
substrate concentrations used. Data from
Lipscomb et al. (52) imply that more than
one enzyme is involved in TCOH formation
in mouse liver. The increase in reaction rate is
not observed at substrate concentrations
above 0.5 mM (concentrations not likely to
be observed in vivo). Analysis of kinetics data
led Lipscomb et al. (52) to conclude that at
higher substrate concentrations, a second,
lower affinity enzyme becomes largely responsible for CH reduction. Similar kinetics, however, could not be demonstrated in rat or
human liver cytosol.
Formation of trichloroacetate and
dichloroacetate. TCA is produced by oxidation
of either CH or TCOH. Oxidation of CH is
believed to be catalyzed by an aldehyde oxidase, whereas oxidation of TCOH is catalyzed by P450, with CYP2E1 likely being
the predominant isoform involved (49). As
mentioned above, metabolism of CH to
TCOH or TCA is slow in mouse lung, leading to accumulation of CH within the Clara
cells. Overall, however, the lung plays a quantitatively minor role in CH metabolism. The
subsequent reactions of CH are rapid in liver,
leading to production of TCOH and TCA.
Kinetics and time course studies in mice, rats,
and dogs of TCE metabolism to CH, TCA,
and TCOH are consistent with TCA being
derived from oxidation of both CH and
TCOH (28,53-55).
Lipscomb et al. (52) reported marked differences in CH metabolism to TCOH and
TCA in liver and blood of rats, mice, and
humans. Determination of kinetic parameters
at physiologically obtainable concentrations of
CH (i.e., in the range of 50 gM) showed that
CH is cleared from human blood through
hepatic metabolism at approximately 60% of
the rate as in rodents. Hence, these data suggest that higher amounts of CH will be present in human blood and tissues than in those
of rodents after a given exposure to TCE.
There has been considerable controversy
and uncertainty about the sources and
amounts of DCA formation, particularly in
humans. Bull and colleagues (54,56,57)
reported detection of DCA in urine of both
rats and mice treated with TCA and in blood
of mice treated with TCE. However, problems were reported with analytical methodologies that led to artifactual overestimations of
DCA formation (58). The AUC for DCA following TCE administration to mice exceeded
that predicted from the formation of TCA
from TCE. Although other pathways for
DCA formation have been proposed and may
occur, it appears that in the presence of strong
acids, some of the TCA that is in whole blood
can undergo nonenzymatic conversion to
DCA, thus leading to overestimation of DCA
formation. In a subsequent study, Bull and
colleagues (55) reassessed formation of
metabolites of TCE in blood from rats, mice,
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
and dogs, and found measurable levels of
DCA only in blood from mice. Similarly, it is
unclear whether DCA is produced in humans
under normal circumstances. Henderson et al.
(59) identified DCA, in addition to TCA,
TCOH, and TCOG, as a metabolite of CH
in children, for whom CH is still used therapeutically as a sedative-hypnotic agent.
Nonetheless, more DCA is formed in mice
than in other species. It is difficult, therefore,
to develop parameters for DCA formation
that can be used in PBPK models for human
health risk assessment. Since both DCA and
TCA produce hepatomegaly and cytomegaly
that may lead to hepatocarcinogenesis (54,56)
and DCA is derived from both TCA and
TCOH, assumptions need to be made until a
more definitive verdict on the formation of
DCA in humans is made. However, both
DCA and TCA are unequivocally hepatocarcinogenic, irrespective of their mechanism or
mode of action (54).
An errata was published based on the
studies of Templin et al. (57) and Abbas et
al. (46) concerning artifacts in the determination of DCA. The bottom line is that the
probable DCA concentrations in biological
fluids are lower than those reported because
of difficulties in analysis.
Formation of other oxidative endproducts.
An additional problem with detection of DCA
in biological samples, particularly tissue from
liver, is the subsequent metabolism of DCA to
other species, such as OX, CAA, glycolic acid,
and glyoxylic acid (GLX). DCA has a much
shorter half-life than TCA in rats and mice
(56), consistent with rapid metabolism or
excretion. Lipscomb et al. (60) described
metabolism of DCA in liver cytosol from male
B6C3F, mice and Fischer 344 (F344) rats that
was dependent on reduced pyridine
nucleotides and GSH but was oxygen independent. Stacpoole and colleagues (61)
showed that liver cytosol from both rats and
humans catalyzes the GSH-dependent conversion of DCA to GLX. The reaction is
NAD(P)H-independent and exhibits a Km for
GSH of only 75 iM. It is unclear whether a
GSH S-transferase (GST) isozyme is involved
in the reaction.
Liver microsomes and mitochondria
exhibit insignificant activity of DCA degradation (60). Hence, although dehalogenation of
DCA to CAA may occur by P450-mediated
catalysis, the major pathway(s) for hepatic
DCA degradation appears to be P450-independent. In fact, Anders and Tong (62)
recently showed that a newly described isoform
of GST, GST4, is involved in the GSHdependent oxygenation of DCA to form GLX.
The significance of these metabolites of
DCA in TCE-induced toxicity and carcinogenesis is unclear, but their role is likely to be
quantitatively minor. Little or trace amounts
181
LASH ET AL.
above background of OX, CAA, glycolate,
and GLX are detected in in vivo exposures to
TCE or in in vitro incubations with TCE.
OX is poorly soluble in water, leading to precipitates. Hence, extensive formation of OX
might be expected to lead to formation of
stones in the urinary tract.
Tissue Distribution of Oxidative
Metabolism of TCE
Relative rates of oxidative metabolism of
TCE in key target organs are important in
understanding how these tissue-dependent
differences affect toxicity. Besides P450,
which catalyzes the initial step of TCE
metabolism, tissue-dependent differences in
secondary enzymes that act on metabolites of
TCE, such as alcohol dehydrogenase and
UDP-glucuronosyltransferase, may be important in that differences in these activities relative to P450 may alter the distribution of
metabolites so that one or more metabolites
may accumulate, leading to a pattern of toxicity that does not occur in tissues that have a
different distribution of these activities.
Quantitatively, the liver is the most
important site of oxidative metabolism of
TCE due to the first-pass effect. Regardless of
the route of administration (i.e., dermal,
inhalation, oral), most of the TCE is rapidly
absorbed into the circulation and goes to the
liver. Only in specialized circumstances does
the liver not play a key role or at least some
role in determining overall metabolism and
toxicity.
A situation where differential activities of
the enzymes of the oxidative pathway of TCE
metabolism markedly affect the distribution
of metabolites and the toxicity is that of CH
accumulation in mouse lungs (44,45). Clara
cells of mouse lungs have relatively high P450
activity but have low activities of alcohol
dehydrogenase and UDP-glucuronosyltransferase. Hence, the enzymes that metabolize
CH and TCOH are believed to be present in
low amounts relative to P450, leading to an
accumulation of CH in the cells. Since
TCOH is recovered in significantly greater
amounts and as a higher fraction of total
TCE metabolites in mice than in other
species, UDP-glucuronosyltransferase may be
present at relatively low levels compared to
the other enzymes involved in oxidative
metabolism of TCE, not only in the mouse
lung, but in other mouse tissues as well,
including the liver.
Besides the liver, the kidneys may be
directly exposed to TCE or some of its
metabolites, since they receive 25% of the
cardiac output. While the kidneys contain
P450 activities, induding CYP2E1 (at least in
rodents), most of it is in the proximal tubules
and the total activity in the tissue as a whole
is markedly lower than that in the liver.
182
Nonetheless, renal oxidative metabolism of
TCE occurs, albeit at rates that are 3- to 10fold lower than those in the liver (63), and
may play some role in either nephrotoxicity/
nephrocarcinogenicity or, via renal-hepatic
circulation, in liver injury.
Interorgan metabolism, which includes
enterohepatic and renal-hepatic circulations
of both the parent compound and several of
the major oxidative metabolites, is another
major issue in the development of models of
TCE pharmacokinetics. Interorgan metabolism can also enhance the further biotransformation of TCE metabolites and has
implications for target organ toxicity. For
example, TCOG that is formed in the liver is
excreted into bile, but little fecal excretion is
observed due to extensive enterohepatic circulation. Once TCOG returns to the liver, it
may be hydrolyzed back to TCOH and be
metabolized further to TCA or DCA. In fact,
TCA is the major circulating metabolite of
TCE, likely due to its high affinity for binding to plasma proteins. A biphasic pattern of
TCA concentrations is found in blood in the
mouse (Table 2), consistent with enterohepatic circulation of TCOH. Thus, enterohepatic circulation plays a major role in the
disposition of TCOH and TCOG. For the
oxidative pathway of TCE metabolism, the
major role for renal-hepatic circulation is in
the excretion of metabolites in the urine. The
major metabolites of TCE that are recovered
in urine include TCA and TCOH/TCOG.
Sex- and Species-Dependent
Heterogeneity in Oxidative
Metabolism of TCE
As described in the reviews on mode of action
in the various target organs, marked differences
in susceptibility to TCE-induced toxicity are
seen in males and females of each species and
between species of a given sex. Since toxicity is
directly associated with specific metabolites,
the likely explanation for these differences in
susceptibility are sex- and/or species-dependent
differences in either overall rates of metabolism
or distribution of metabolites among specific
pathways. An understanding of the factors that
determine these susceptibilities is essential for
being able to extrapolate animal data to
humans. An additional point, however, is that
these sex- and species-dependent differences
are often dose dependent in that markedly different types of effects may be seen at high
versus low doses.
Overall rates of TCE metabolism vary
considerably between the various experimental
species that are used in laboratory investigations (i.e., rats, mice, rabbits) and between
these species and humans (64). In practical
terms, more complete documentation and
understanding of sex- and species-dependent
differences in metabolism will enable
refinements in risk assessment and definitions
of acceptable exposure levels. To extrapolate
metabolic data between species in the development of pharmacokinetic models, default allometric scaling procedures are often used.
These involve scaling fluid flows (e.g., blood,
bile, urine) by body weight to the 3/4 power
and scaling rate constants by body weight to
the 1/4 power. Additional uncertainty factors
are applied when there is uncertainty regarding intraspecies sensitivities or when there are
uncertainties in extrapolating from one species
to another. Again, these factors are intended
to account for many processes, not just
metabolism or pharmacokinetics. Whether
these default assumptions are representative of
the actual physiological state is an open question. For example, the activity of various
P450s in humans can vary by up to a factor of
10 among individuals (35,64,65), so that
default assumptions can be significantly different than the actual situation in humans.
However, the observed variations are often
beyond allometric expectations, suggesting
that more complex metabolic and other
differences exist between species.
As described earlier, studies by Prout et al.
(7) showed that blood levels of the TCE
metabolites TCOH, CH, and TCA were
severalfold higher in B6C3F1 mice than in
Osborne-Mendel rats after a single oral dose
of TCE. Larson and Bull (54) similarly
showed that the initial rates of TCE metabolism to TCA were much higher in mice than
in rats, leading to higher TCA concentrations
in blood. Nakajima et al. (42) also showed
that rates of oxidative metabolism of TCE in
liver microsomes of male B6C3F1 mice were
2- to 3-fold higher, depending on dose, than
those from male Wistar rats. Furthermore,
overall content of P450 (nmol P450/mg
microsomal protein) did not differ in the two
species. Rather, the higher rate of TCE
metabolism was due specifically to higher levels of CYP2E1 in mouse liver microsomes. In
general, metabolic rates for most chemicals
are significantly higher in mice than in rats or
other larger animals, including humans.
A comparison of kinetic parameters for
overall P450-catalyzed metabolism of TCE in
liver microsomes from male B6C3F1 mice,
F344 rats, and humans is shown in Table 4.
Several species-dependent differences are
immediately apparent. First, kinetics were
clearly biphasic in liver microsomes from rat
and human but were monophasic in liver
microsomes from mouse. Rates of TCE
oxidative metabolism for the high-affinity
processes were approximately 2- to 2.5-fold
faster than those for the low-affinity processes
in rats and humans. Finally, the ratio of V,,
to Km values, which is a measure of catalytic
efficiency, shows that although rates are lower
than in the other species, the activity in
Environmental Health Perspectives
*
Vol 108, Supplement 2 * May 2000
METABOLISM OF TCE
humans exhibits similar efficiency to that in
rats and mice.
This similarity between rats and humans
regarding P450-dependent metabolism of
TCE may extend to other aspects of the
metabolic process. Both the rat and human
2E family contain only one gene and expression of CYP2E1 in both species is regulated
both transcriptionally and posttranscriptionally (65). In contrast, the rabbit 2E subfamily
comprises two genes. Although rabbit
CYP2E1 appears to be very similar to human
and rat CYP2E1 with respect to substrate
specificity and regulation of expression, rabbit
CYP2E2 is not coordinately controlled with
CYP2E1 and developmental expression of the
two forms differs. Thus, Wrighton and
Stevens (65) and Lipscomb et al. (66) concluded that the rat is a better model than the
rabbit for study of human CYP2E1 expression. The data summarized above, comparing
P450-dependent metabolism of TCE in rats,
mice, and humans, support this conclusion
and indicate further that the rat is a better
model than the mouse for study of oxidative
metabolism of TCE in humans.
Although sex-dependent differences have
been clearly observed in the susceptibility to
TCE-induced toxicity and carcinogenicity,
few studies of sex dependence of TCE metabolism have been conducted. Nakajima et al.
(41) studied sex-, age-, and pregnancyinduced effects on the expression and regulation of CYP2E1 and CYP2C1 1 with regard
to TCE and toluene metabolism in Wistar
rats. They observed no sex-dependent differences in TCE metabolism. The only differences in rates of TCE metabolism to CH that
were observed were an approximately 3-fold
decrease from 3 weeks of age (puberty) to 18
weeks of age (maturity) in both sexes and a
nearly 2-fold decrease in rates in females during pregnancy.
Variations among Humans
Lipscomb et al. (64) observed considerable
variability in the P450-catalyzed oxidation of
TCE in 23 samples of human liver microsomes. Km values for TCE (28.3 ± 12.9 pM;
mean ± SD, n = 23) varied over a more than a
fourfold concentration range (range =
12.6-55.7 pM) and were not normally distributed. Vmax values for CH formation
(1,589 ± 840 pmol/min/mg; mean ± SD, n =
23) also showed considerable variability, with
samples from individual human livers exhibiting a 7-fold range of values (range =
490-3,455 pmol/min/mg). In their analysis
of the kinetics of TCE metabolism, individual samples seemed to cluster into three
groups, having Km values (pM TCE, mean ±
SD) of 16.7 ± 2.5 (n = 10), 30.9 ± 3.3 (n=
9), and 51.1 ± 3.8 (n = 4). Within each
group, there were no patterns with regard to
the ethnic group or sex of the donor from
whom the livers were isolated, and Vm,, values in each group exhibited the same degree
of variability and span of values as the entire
sample pool. The Km value in females (21.9 ±
3.5 pM; n = 10) were significantly lower than
that in males (33.1 ± 3.5 pM; n = 13).
Analysis of the activity of three specific
P450 isoforms that are known to catalyze
TCE metabolism in human liver microsome
samples from the three groups revealed some
patterns (Table 5). CYP1A2 activity was significantly lower in the low-Km group than in
the other two groups, and CYP2E1 activity
Table 4. Apparent kinetic constants for total oxidative metabolite formation from TCE.8
Species
Mouse
Male
Female
Rat
Male
(High affinity)
(Low affinity)
Female
(High affinity)
(Low affinity)
Human
Male
(High affinity)
(Low affinity)
Female
(High affinity)
(Low affinity)
Vmax
(nmol/mg per min)
Km (mM)
Vmam/Km
n
8.60 ± 4.50
26.1 ± 7.3
0.38 ± 0.41
0.16 ± 0.03
42.0 ± 28.5
163 ± 37
5
3
0.96 ± 0.65
2.48 ± 0.97
0.072 ± 0.082
0.482 ± 0.104
23.8 ± 20.6
5.3 ± 2.2
5
2.91 ± 0.71
4.31 ± 0.31
0.042 ± 0.021
0.111 ± 0.027
80.0 ± 33.9
40.1 ± 7.1
3
0.52 ± 0.17
0.93 ± 0.17
0.012 ± 0.003
0.093 ± 0.026
48.0 ± 23.1
10.7 ± 3.9
3
3
15.3 ± 10.1
6.8 ± 5.6
aOxidative metabolism of TCE was measured by incubation of microsomes with 10 pIM-2 mM TCE in the presence of 2 mM NADPH
for 10 min at 37°C and pH 7.4. The following metabolites (assay limits of detection in parentheses) were measured after derivatization
with pentafluorobenzyl bromide and gas chromatography with electron capture detection: TCA (1 ppm), DCA 10.025 ppm), CM (0.025
ppm), OX (10 ppm). TCOH (0.001 ppm) was detected without any derivatization by gas chromatography with electron capture detection. CH (0.75 ppm) and GLX (0.1 ppm) were detected by HPLC after derivatization with 2,4-dinitrophenyl hydrazine. Data are means ±
SD of measurements from the indicated number of experiments and were reported in Elfarra et al. (70).
0.33 ± 0.15
0.72 ± 0.60
Environmental Health Perspectives
*
Vol 108,
0.026 ± 0.017
0.160 ± 0.162
Supplement
2
*
May 2000
was significantly higher in the high-Km group
than in the two other groups, but no differences were observed for CYP3A4 activity
among the three groups.
Lipscomb et al. (64) also found that
CYP2E1 was the primary isoform responsible
for TCE metabolism, accounting for > 60%
of total microsomal metabolism. These results
indicate that the capacity of humans to
metabolize TCE will vary considerably and
that factors that alter P450 activity, in particular CYP2E1 activity, can alter TCE metabolism and hence, susceptibility to TCE-induced
toxicity. Nakajima et al. (41) also observed
significant variation in oxidative metabolism
of TCE as a function of physiological state
(see above).
Comparative in Vitro
Evaluation of Cytochrome
P450-Dependent
Metabolism of TCE
This section illustrates an integrated in vitro
approach to evaluation of TCE metabolism
by the P450 pathway with the goal of elucidation of the kinetics of metabolism of TCE
to CH, TCOH, TCA, and DCA in the rat,
mouse, and human. These data are based on
a series of studies conducted at the Armstrong
Laboratory (Toxicology Division at WrightPatterson AFB, Ohio) (11,52,60,64,67,68).
Data indicate a) that human hepatic microsomes possess less activity toward TCE than
either rat or mouse hepatic microsomes, b)
that TCOH formation proceeds at higher
rates than TCA formation at low CH concentrations in all three species, and c) that
appreciable levels of TCOH and TCA are
formed from CH in the blood of all three
species in vitro. The formation of DCA has
been questioned for years, and no mechanism
for its formation has been fully validated.
DCA has not been identified in liver preparations (i.e., microsomes, homogenates, tissue
slices) from mice or humans exposed to TCE,
CH, or TCOH in vitro. Cultured gut
microflora from mouse cecum produce DCA
from TCA in a dose- and time-dependent
Table 5. Evaluation of form-selective cytochrome P450
metabolism in human liver microsomes expressing different Km values for TCE metabolism.a
CYP form
CYP3A4
CYP2E1
CYP1 A2
Group
±
2.65 ± 2.661
520 295
Low-Km 241 ± 186
820 ± 372
2.92 ± 2.78b
Mid-Km 545 ± 200
1.78 ± 1.08b
High-Km 806 ± 442 1,317 ± 592
&Activities of CYP1A2, CYP2E1, and CYP3A4 were measured
with phenacetin, chlorzoxazone, and testosterone as substrates,
respectively. Data are means ± SD of values from 10, 9, and 4
samples for the low-, mid-, and high-Km group, respectively.
Data within each isozyme assay with the same letter are not
significantly different from one another (p< 0.05) by the KruskalWallis one-way ANOVA.
183
LASH ET AL.
As described above, evaluation of the data concentrations of TCE in the human. To
from individual human liver samples revealed determine whether CYP2E1 was the preconsiderable variation, with Km values rang- dominant P450 form responsible for TCE
ing from 16 to 56 luM and V,. values rang- metabolism in a sample of 27 human hepatic
ing from 490 to 3,455 pmol/min/mg, microsomes, the activity of a known subalthough there was no correlation between strate for CYP2E1, chlorzoxazone (CZX)
Km and Vn values among individuals.
(71,72), was compared with TCE metabolic
TCE was an effective inhibitor of activity (data not shown). Results indicate
CYP2E1, as measured by p-nitrophenol that TCE metabolism is significantly correhydroxylase activity (data not shown). Three lated (r2 = 0.51, p < 0.05) with CYP2E1
individual samples of human liver micro- activity in humans (11). These data provide
somes, representing individuals from the low- further support for the involvement of
mid-, and high-Km groups (see above), exhib- CYP2E1 in TCE metabolism in the human.
The high degree of conservation of
ited concentration-dependent inhibition of
CYP2E1 activity. The degree of inhibition CYP2E1 across mammalian species and the
appeared to parallel the Km of the microsomes high degree to which it is expressed in rodent
Chloral Hydate Formation
for TCE metabolism, suggesting that liver, compared to human liver, make the
Concentrations of TCE reported here, includ- CYP2E1 makes a larger contribution to TCE determination of inherent CYP2E1 activity
ing Km values, represent the concentration of metabolism in the high-Km sample than in and the assessment of factors that modify
TCE in the liquid phase of the incubation sys- either of the other two samples. Kinetic CYP2E1 expression in the human important
tem. Gas chromatographic quantitation of analysis of the inhibition of p-nitrophenol considerations in population risk assessments.
CH and TCOH in the incubation mixture hydroxylase activity by TCE in mouse liver To determine the variability of CYP2E1 and
was accomplished by analysis of the ethyl microsomes showed the inhibition to be com- gain an indication of the potential variability
acetate extract using modifications of the petitive (data not shown), as would be of TCE metabolism in the human, microsomethod of Maiorino et al. (69). Neither TCA expected if CYP2E1 metabolizes TCE.
mal metabolism of CZX and the degree of
nor DCA was detected (detection limit = 1.5
To determine the extent to which expression of total P450 content was examnmol/mL) under these conditions. Because CYP2E1 metabolized TCE in humans, ined in 54 human hepatic microsome samples
CH is further metabolized to TCOH, CH experiments were designed using equivalent (Figure 3). Data indicate that there is an
and TCOH that were quantitated in microso- content of human CYP2E1, whether derived approximate 5-fold variability in the expresmal incubations dosed with TCE were com- from human CYP2E1 gene transfected and sion of CYP2E1-dependent CZX metabolism
bined to give an estimate of TCE metabolism. expressed in a lymphoblastoid cell line or in this population. Because of the degree of
Significant differences between the three authentic human hepatic microsomes. The correlation of TCE metabolism with
species were found in the Michaelis-Menten results (Figure 2) demonstrate that human CYP2E1-dependent activity, it may be reakinetic parameters evaluated for CH forma- CYP2E1 expressed in a lymphoblastoid cell sonably predicted that this degree of variabiltion from TCE. Overall kinetic parameters line metabolizes approximately 75% as much ity in TCE metabolism will be seen in the
for TCE metabolism in rat, mouse, and TCE as an equivalent amount of authentic human population. Such variation and facpooled human liver microsomes (Table 6) human liver microsomes. These data indicate tors that induce the activity of CYP2E1 may
demonstrate a lower rate in the human than that CYP2E1 is the major enzyme responsi- define segments of the population that may
either the rat (1:3.3) or the mouse (1:3.8). ble for the microsomal oxidation of low be at a higher risk than would be otherwise
Evaluation of these kinetic data over a wide
anticipated (73).
range of TCE concentrations by construction
The data also indicate that other enzymes
of Eadie-Hofstee plots allowed for separation
may metabolize TCE in the human. Because
of data from rat liver microsomes into three
a high fraction of P450 is accounted for by
kinetically distinct components, consistent
I
with Nakajima et al. (39). The three components had Km values (1iM TCE) of 17, 114,
and 909 pM and V,,, values (pmol/min/mg)
[.01
of 818, 1,275, and 2,797. A similar analysis
of data from mouse liver microsomes revealed
homogeneity, with only one kinetically disI4
tinct component. These results agree with
0.001
those described above from Elfarra et al. (70).
16pMTCE
1IN MTCE
manner. The transport of TCA from liver
into blood or bile may influence net DCA
formation; TCA in blood may be cleared into
the urine, whereas TCA in bile enters the
small intestine and may drive TCA production in the gut. However, in mice treated
with antibiotics to eliminate gut microbes,
plasma DCA concentrations following TCE
treatment were only minimally affected, suggesting that non-gut mechanisms of DCA
formation also exist. In summary, these in
vitro studies indicate a lower rate of TCE
metabolism and hence, a lower rate of TCA,
TCOH, and DCA formation in the human
as compared with the mouse and rat.
Table 6. Overall kinetics of TCE metabolism to CH and
TCOH.a
Species
Rat
Mouse
Human
MS-HI KMi
Km (pM TCE)
55
35
25
Vmax(pmol/min/mg)
4,826
5,425
1,440
'Metabolism of TCE to CH and TCOH was measured in rat,
mouse, and human liver microsomes by gas chromatography.
Microsomes were pooled from 5 rodent and 7 human liver samples and were incubated with from 7.5 to 1,000 pM TCE for
either 10 (rodent) or 30 (human) min.
184
MS-UW "
X
=I:~
1A2
IAI
3A4
Figure 2. Metabolism of TCE by human liver microsomes
and genetically-expressed P450 forms. Assays contained
1.0 mg liver microsomal protein/mL [which has been
shown to contain 22 pmol of CYP2E1/mg protein (73)1 or
22 pmol/mL of CYP2E1, 42 pmol/mL of CYP1A, or 96
pmol/mL of CYP3A4 expressed in microsomes from
genetically engineered lymphoblastoid cells. Samples
were incubated with 16 or 100 pM TCE and CH formation
was measured after 30 min by gas chromatography.
Figure 3. Distribution of CZX metabolism in a sample
population of human liver microsomes. Histogram
depicting the frequency distribution of metabolism of
CZX, a marker substrate for CYP2E1, in individual human
liver microsome samples. The results reveal a population with a normal distribution.
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
METABOLISM OF TCE
CYP3A4 and CYP1A2 (72), the effect on
TCE metabolism of known substrates for
these forms was evaluated. a-Napthoflavone
(a CYPIA substrate) and ketoconazole (a
CYP3A substrate) did not inhibit TCE
metabolism when assessed at TCE concentrations near the Km value (64) but reduced
Vmax values by 30 and 18%, respectively,
when evaluated at TCE concentrations 4-fold
above the Km value. These data indicate that
CYP1A2 and CYP3A4 also metabolize TCE
in the human but that their contribution to
overall TCE metabolism is low compared to
that of CYP2E1.
Trichloroethanol and Trichloroacetic
Acid Formation
TCOH is derived from CH. Species-dependent differences in the metabolism of CH
were evaluated in a clarified liver homogenate
(52). This preparation was used because various enzymatic activities capable of metabolizing CH are distributed in several subcellular
compartments (74). NADH is the best
nicotinamide cofactor for the stimulation of
TCOH production in vitro in all three
species. However, in human liver, NADPH
also significantly stimulated TCOH production. For each of the cofactors tested, the
addition of cofactor resulted in more TCOH
production than in controls with no cofactor.
The final evaluation of CH metabolism in
the liver involved the determination of cofactor kinetics in the formation of TCOH. Data
revealed that Km* values (the concentration of
cofactor that produced half-maximal metabolic rates) for these reactions in rodent liver
approximated the levels of pyridine
nucleotide cofactors reported for rodent liver
(75). Km* values (mM NADH) for TCOH
formation were 0.91 and 0.36 mM for the rat
and mouse, respectively.
An evaluation of clearance (VmJKj) of
CH to TCA and TCOH was done to compare the net conversion of CH to these
metabolites. The results in Table 7 indicate
that clearance of CH to TCOH is much
higher (10- to 200-fold) than clearance of
CH to TCA in these three species. Clearance
of CH to TCOH by the mouse high-affinity
enzyme was highest, but this enzyme becomes
inhibited at CH concentrations above 0.5
mM. Clearance of CH to TCOH in the
human was approximately half that observed
in rats and approximately twice that observed
with the mouse low-affinity enzyme.
Clearance of CH to TCA in the mouse and
human were similar, whereas it was approximately 10-fold lower in the rat. These data
indicate that TCOH formation will predominate over TCA formation in the liver of these
three species. The kinetic evaluation of
TCOH production also revealed that the
mouse may have two separate enzymes
Table 7. Kinetics of TCOH and TCA formation from CH.a
TCOH
Vmax
Km
24.3
0.52
Rat
Mouse
6.3
0.12
High affinity
6.1
Low affinity
0.51
34.7
1.34
Human
TCA
VmayIKm
Km
Vmax
VmaVKm
46.7
16.4
3.5
4.0
10.6
0.24
3.03
23.9
65.2
2.73
52.5
12.0
25.9
aHomogenate from the liver of 4 individuals from each species was centrifuged at 700xg and the supernatants obtained were pooled.
Km is presented as mM CH in solution that contained 0.9 mM NADH and 0.9 mM NAD+ for experiments for TCOH and TCA formation,
respectively. Vmax is reported as nmol/min/mg 700xg supernatant protein. The mouse high-affinity enzyme responsible for TCOH formation was inhibited at CH concentrations > 0.57 mM.
responsible for formation of TCOH from
CH in liver, whereas the rat and human
apparently possess a single enzyme. The highaffinity form in the mouse is inhibited at CH
concentrations above 0.5 mM (the nature of
the inhibition is unknown), and the lowaffinity form becomes saturated at concentrations of CH above 1.0 mM. This
bifunctional metabolism is also seen in mouse
hepatic cytosol (76). These results indicate
that at least one mouse cytosolic enzyme
responsible for TCOH formation becomes
inhibited at high CH concentrations.
TCA production from CH by mouse, rat
and human liver supernatant was best stimulated by NAD+. Of the reduced cofactors,
only NADPH stimulated TCA production
above that of control, and this occurred only
in the mouse. These results (particularly the
degree to which mouse liver TCA production
is stimulated by both NAD+ and NADP+)
indicate that there are species-related differences in hepatic TCA formation. The apparent kinetic constants for the formation of
TCA from CH in the rat, mouse and human
in the presence of NAD+ indicate a much
lower Km value in the mouse, indicating that
under equivalent CH concentrations, TCA
formation will proceed at a more rapid rate in
mouse liver (Table 7). It is doubtful whether
CH will attain concentrations in human liver
to drive metabolism near the theoretical maximal value. Km values (CH) for TCA are
much higher than those required for TCOH
production, indicating that low concentrations of CH will favor the production of
TCOH. Km* values (mM NAD+) for TCA
production were 0.13 and 0.14 mM for the
rat and mouse, respectively.
The kinetic parameters indicate that at
low hepatic concentrations of CH (below 0.5
mM), TCOH will be the preferred hepatic
metabolite. As shown in Table 8, the predicted ratio of TCOH:TCA is 10 to 20,
depending on species. These predictions agree
well with the observed urinary elimination of
TCOH and TCA in mice and rats exposed to
200 mg TCE/kg (53,54) and with initial
(0-1 hr) plasma levels of metabolites observed
in CH-exposed humans, where the levels of
TCOH were slightly higher than plasma levels
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
Table 8. Predicted rates of hepatic metabolite formation
at 50 pM chloral hydrate.a
Metabolite
TCA
TCOH
Species
0.012
2.13
Rat
Mouse
0.149
2.35
0.136
Human
1.25
'Metabolite (TCOH, TCA) formation is presented as nmol/min/mg
700 x g supernatant from liver homogenates of the indicated
species. Metabolic rates were calculated from the MichaelisMenten equation using the parameters obtained in Table 7.
of TCA (51,77). There is also evidence for
the oxidation of TCOH to TCA (77,78). If
TCOH is an obligate intermediate in the formation of TCA from CH, the accumulation
of TCOH in blood and liver exposed to CH
in vitro implies that the initial conversion of
CH to TCOH is not rate limiting in the
formation of TCA from CH.
Chioral Hydrate Metabolism by Blood
The conversion of CH to TCA and TCOH
was examined in blood of the rat, mouse, and
human to confirm and extend the findings of
an earlier report (51), which demonstrated
that significant amounts of TCOH and limited amounts of TCA were formed by blood
in vitro. Results from experiments with lysed
whole blood (no exogenous cofactor added)
indicate that much more TCOH than TCA is
produced (Table 9). Human blood produced
less TCOH (on a milliliter-for-milliliter basis)
than did either rat or mouse blood. However,
TCA production in lysed human blood was
significantly greater than TCA production by
rat blood but only slightly higher than that by
mouse blood. The further examination of
lysed erythrocytes and plasma of the human
indicate that erythrocytes were the sole site of
blood TCA production (best stimulated by
NADP+). Plasma formed 4- to 5-fold as much
TCOH from CH as did an equal quantity of
packed erythrocytes, indicating that plasma is
the primary site of TCOH production (best
stimulated by NAD+) by blood. These data
underscore the need to evaluate the organ-specific, presystemic, extrahepatic formation and
metabolism of CH derived from a TCE exposure and to fully describe CH metabolism in
the blood compartment. Evaluation of the
distribution of CH from portals of entry into
185
LASH ET AL.
decreased gut DCA content compared with
animals exposed to TCE without antibiotic.
DCA present in circulating plasma was only
Human
Rat
Mouse
slightly diminished in antibiotic-treated mice
46.7 ± 1.0b
20.5 ± 0.7c
TCOH 45.4 ± 4.9b
the source of DCA in
TCA 0.135 ± 0.020d 0.208 ± 0.030e 0.269 ± 0.030 (68). Therefore,
plasma is still unknown.
aResults are presented as nmol product formed in a 400-pL
lysate (containing 360 pl whole blood + 100 pL CH) over 30 min Dichloroacetic Acid Metabolism
and are means ± SD of measurements from 3 experiments.
Means with the same superscript letter are not significantly dif- DCA degradation occurs primarily in rat
ferent from one another at p < 0.05.
hepatic cytosol (60). Recent studies have
revealed similarities between kinetic constants
blood and a kinetic examination of blood- for hepatic cytosolic DCA degradation in the
catalyzed CH metabolism will further the mouse and rat in vitro and marked differences
success of species-dependent examination of between the kinetic constants in the human.
CH metabolism in vivo and can be used to These studies reveal Km values of 350, 280,
refine the PBPK models for both TCE and and 71 pM and Vmaxvalues of 13.1, 11.6,
CH. Kinetic data would verify the impor- and 0.37 nmol/min/mg protein for the
tance of hepatic CH metabolism in the over- mouse, rat and human, respectively (60).
all disposition of toxicologically relevant TCE Evaluation of clearance values (VmaxKm) indimetabolites.
cates that human liver cytosol degrades DCA
less efficiently than either rat or mouse liver
Table 9. Metabolism of CH to TCOH and TCA by lysed,
whole blood.a
Dichloroacetic Acid Formation
firom Trichoroacetic Acid
cytosol.
DCA is a TCE metabolite that is readily
identified in rodents but is generally not identifiable in humans. DCA has not been
detected under either aerobic or anaerobic
incubations of hepatic fractions with TCE.
While it has been impossible to demonstrate
the hepatic formation of DCA from TCE in
vitro, it has been demonstrated that the
dechlorination of TCA to DCA is catalyzed
by gut contents (ingested food and bacteria)
of the rat and mouse. Microflora cultured
from the cecum of the B6C3F1 mouse dechlorinate TCA to DCA in a dose- and timedependent manner (Figure 4) (67). To
confirm these in vitro observations in vivo,
B6C3F1 mice were treated with antibiotics to
virtually deplete the mouse gut of microflora.
Antibiotic treatment resulted in dramatically
increased gut TCA content and concurrently
2J
41
-& - 3-hr
4-hr
/ /
10a
for TCE metabolism
normally
distributed
sample of human hepatic
microsome preparations; Km values were
divisible into three statistically distinct
Metabolic step
TCE to CHa
Km (pM TCE)
Vmax(nmol/min/mg microsomal protein)
CH to TCOHb
Km (mM CH)
-0- 1-hr
-0- 2-hr
15 *
l
mouse. Furthermore, in vitro kinetic parameters
were not
in a
23
Table 10. Comparison of metabolic parameters for the oxidative metabolism of TCE determined in vitro.
-
20-
Species-Dependent Differences in
P450-Dependent Metabolism of TCE:
Interpreation of Findings for Human
Health Risk Assessment
Several steps of the TCE metabolic pathway
were evaluated in vitro and striking speciesdependent differences were demonstrated.
Kinetic parameters for several of the steps in
the oxidative pathway of TCE metabolism
are summarized in Table 10. All available in
vitro data indicate that the human is less
capable to metabolize TCE and CH than the
rodent. The rate of CH formation in the
human is approximately 20% of that of the
Vmax(nmol/min/mg supernatant protein)
VmaxVKm
10
CH to TCAb
15
0
200
400
600
800
[TCA], ,M
Figure 4. Time and concentration dependence of DCA
formation by anaerobic mouse gut microflora cultures.
Cultures of bacteria from mouse gut were incubated (108
cells/mL) with the indicated concentrations of TCA for
up to 4 hr.
186
CYP2E1 is the form identified as the
major determinant of TCE metabolism in the
human, and the form is well conserved across
species but is expressed to a higher extent in
rodents than humans. CYP2E1 exhibits a Km
value for TCE of approximately 20 gM. The
differential expression of CYP2E1 across
species may in part explain the higher TCE
metabolism seen in rodents than in humans
in vitro. The examination of several sets of in
vitro data also indicates that CYPIA and
CYP3A forms metabolize TCE at concentrations above the Km value for CYP2E1. The
Km value for TCE metabolism in microsomes
of female humans is significantly lower than
that in males, but V,", values were not significantly different (64). Factors that alter the
expression or activity of CYP2E1 in the
human should be incorporated into estimates
of TCE metabolism in vivo. TCOH and
TCA formation by components of mouse,
rat, and human blood was demonstrated and
TCOH was identified as the major bloodderived CH metabolite. The metabolism of
CH to TCOH and TCA is efficiently catalyzed by liver homogenates from all three
species, and TCOH formation predominated
over TCA formation under all conditions
evaluated in vitro. Km values for TCOH formation are approximately 20-fold lower than
Km values for TCA formation, which drives
the formation of TCOH at higher rates than
TCA formation at low CH concentrations.
Km values for cofactor involvement in hepatic
CH metabolism were shown to approximate
cofactor concentrations found in vivo. These
data indicate that significant changes in either
cofactor level or redox status of the liver may
impact the extent of CH metabolism and/or
the ratio of TCOR:TCA produced. Such an
impact has been demonstrated in the isolated
perfused rat liver (79,80).
groups.
Mouse
35.4
5,425
0.12 (low affinity)
0.51 (high affinity)
6.3 (low affinity)
6.1 (high affinity)
Human
55.5
4826
24.6
1440
0.52
1.34
24.3
34.7
52.5 (low affinity)
46.7
25.9
3.5
10.6
16.4
4.0
0.24
23.9
65.2
12.0 (high affinity)
Km(mM CH)
Vinax(nmol/min/mg supernatant protein)
Vma,/Km
DCA degradation
Km (mM DCA)
Vmax(nmol/min/mg cytosolic protein)
Vmax/Km
Rat
3.03
0.350c
13.1
37.4
2.73
0.280d
0.071c
11.6
0.37
41.4
5.2
'Data reported in Lipscomb et al. (8). hData reported in Lipscomb et al. (52). cData reported in Lipscomb et al. (165). dData reported in
Lipscomb et al. (61).
Environmental Health Perspectives * Vol 108, Supplement 2
*
May 2000
METABOLISM OF TCE
DCA formation was not demonstrated in
any tissue fraction evaluated in vitro. It was
shown that gut contents of mice and rats
dechlorinate TCA to DCA. Additional studies
in antibiotic-treated mice in vivo revealed only
slight depressions of plasma DCA levels following oral administration of TCE, indicating
an additional, nongut source of DCA production. Because of the absence of DCA in samples from humans exposed to TCE, the
degradation of DCA in vitro was evaluated.
Human hepatic cytosol was less effective at
degrading DCA than cytosol of rats or mice.
These findings may explain the lack of demonstration of DCA in human blood following
TCE exposure. Based on the assumption of
equal rates of DCA formation across species,
these results indicate that clearance of DCA by
liver in the human does not account for the
lack of finding DCA. Because TCA is also
formed by components ofblood, because TCA
is more efficiently bound to plasma constituent
proteins in the mouse than in the human,
because binding of TCA by plasma proteins
influences the distribution of TCA from blood
to liver, and because TCA is equally well (if
not more efficiently) formed in the liver of the
mouse than of the human, it may follow that
the human liver is less exposed to TCA than
the mouse liver. If hepatic TCA concentrations
drive biliary elimination of TCA, and TCA
accumulation in the gut drives DCA formation, then it may be reasoned that DCA formation in the human is likely to occur at a
much lower rate than in the mouse. However,
gut is not the sole source of DCA formation.
DCA levels in plasma were diminished but not
eradicated in the antibiotic-treated mouse.
These and other data complicate the inclusion
of DCA in human health risk assessments of
CH and TCE. The usefulness of in vitro data
in the evaluation of the toxicity of TCE and
human health risk assessment should be carefully weighed. While the traditional strength of
in vitro data lies in mechanistic evaluations and
the conditions employed do not reflect the
total in vivo system, the accurate extrapolation
of these (and other) in vitro data will yield
important results. Precedent has been set for
the use of in vitro data in PBPK models (e.g.,
81). A careful extrapolation of in vitro kinetic
data from rat and mouse liver, the evaluation
of pulmonary metabolism of TCE to CH,
blood-mediated metabolism of CH to TCA,
and plasma-binding characteristics of TCA will
greatly aid the construction and validation of
these models, lending credibility to the similar
model constructed for the human (11).
Evaluation of model effectiveness can be
accomplished by comparing kinetic predictions
with currently available human data. This in
vitro approach will contribute to a valid,
science-based, and technologically complex
human health risk assessment for TCE.
Glutathione-Dependent
Metabolism of TCE
This section describes the GSH conjugation
(mercapturic acid) pathway for TCE. In classic drug metabolism textbooks, GSH conjugation has typically been described as a
detoxication route for reactive electrophiles.
For the majority of chemicals that are conjugated with GSH, this is a correct functional
characterization. It became readily apparent
several years ago, however, that GSH conjugation plays a bioactivation role for several types
of drugs, particularly halogenated alkanes and
alkenes. Although conjugation with GSH is
the initial step in the pathway, the critical step
for formation of a cytotoxic and/or carcinogenic metabolite occurs after processing to the
cysteine conjugate. A major issue in the study
of the conjugation of TCE with GSH and the
toxicological consequences of this pathway is
how to accurately measure flux through the
pathway so that its importance relative to the
P450 pathway can be assessed.
Desrption of GSH-Dependent
Pathways of TCE Metabolism
GSH conjugation of TCE: formation of
S-(1,2-dichlorovinyl)glutathione (DCVG). As
shown in Figure 1, the other possible fate of
TCE besides oxidative metabolism is conjugation with GSH, which is catalyzed by the
GSTs. GSTs, like P450s, comprise a family
of isoforms. With the exception of the microsomal GST isoform, all other isoforms are
cytosolic. The primacy of the liver as the site
of GSH conjugation is highlighted by the
observation that GST protein can comprise as
much as 5% of the total cytosolic protein in
rat or human liver. However, GSTs are present in most tissues, including the kidneys, so
that this initial step in the pathway does not
necessarily have to occur in the liver but can
also occur in the target organ.
The cysteine conjugate of TCE, S-(1,2dichlorovinyl)-L-cysteine (DCVC), was discovered in a biological system nearly 40 years
ago (82,83) when it was observed in soybean
meal that had been extracted with TCE.
DCVC was identified as the etiologic agent
that caused nephrotoxicity and aplastic anemia in cows and only nephrotoxicity in several other mammalian species. For many
years, DCVC was seen as a model compound
that arose purely chemically rather than biochemically. An in vivo role for GSH conjugation in the metabolism of TCE was not
appreciated until Dekant et al. (84) showed
that the mercapturate of TCE, N-acetyl-S-
(1,2-dichlorovinyl)-L-cysteine (NAcDCVC),
recovered in the urine of rats that had
been exposed to TCE and Anders and colleagues (85,86) synthesized DCVG and
demonstrated that it was nephrotoxic to rats
was
Environmental Health Perspectives * Vol 108, Supplement 2 * May
2000
and that it was metabolized to DCVC before
exerting its toxicity. This suggested that a
portion of TCE underwent GSH conjugation in vivo with subsequent processing to
the cysteine conjugate followed by N-acetylation and urinary excretion of the mercapturate. Dekant et al. (87) subsequently
showed that TCE was metabolized in rats to
DCVG both in vivo and in isolated liver
microsomes: After administration of TCE in
corn oil by oral gavage, DCVG was recovered in the bile and NAcDCVC was recovered in the urine; DCVG formation was
confirmed in rat liver microsomes incubated
with TCE and GSH.
In a human TCE exposure study (88),
healthy human volunteers (11 males, 10
females) were exposed for 4 hr by inhalation
to either 100 ppm TCE (8 males, 8 females)
or 50 or 60 ppm TCE (3 males, 2 females),
and blood and urine samples were obtained
at various time points both before and after
exposure for up to several days. With respect
to the current discussion of GSH-dependent
metabolism of TCE, the most significant
finding of these studies was that DCVG was
detected in blood within 30 min of the end
of the 4-hr TCE exposure period and its
presence persisted for up to 12 hr. Both
dose- and sex-dependent differences in
DCVG levels in blood were detected and are
summarized in Table 11. DCVG formation
was clearly higher in males at both the
higher and lower doses of TCE, consistent
with the greater susceptibility of males to
renal tumors from TCE [see review on
"Mode of Action for Kidney Tumorigenesis"
in this monograph (89)]. These data, therefore, demonstrate the function of the GSH
conjugation pathway in humans. As discussed below, this is only the initial step in
the generation of nephrotoxic species and
does not directly correlate with it because
subsequent detoxication reactions, such as
mercapturate formation, can still occur.
GSH conjugation of TCE occurs at rates
that are generally severalfold slower than the
P450-catalyzed oxidation reactions. As mentioned above and discussed below in the section on the relative roles of the oxidative and
GSH conjugation pathways in TCE-induced
toxicity and carcinogenesis, there are several
unresolved issues related to the importance
of GSH conjugation in risk assessment for
TCE. Rates of GSH conjugation of TCE are
discussed below in more detail in the sections
on tissue distribution and sex and species
dependence.
Metabolism of DCVG to DCVC.
DCVG, like other GSH conjugates, is
processed by y-glutamyltransferase (GGT)
to the cysteinylglycine conjugate S-(1,2-
dichlorovinyl)-L-cysteinylglycine (DCVCG)
and then by various membrane-bound
187
LASH ET AL.
Table 11. Formation of DCVG in blood from human volunteers exposed to TCE.a
Maximal DCVG recovered (time after exposure)
Males
Females
50/60 ppm TCE
nmol DCVG/ml blood
16.2 ± 8.1 (0.5 hr)
2.13 (1 hr)
pmol DCVG/mg protein
102 ± 81 (3 hr)
16.5 (1 hr)
100 ppm TCE
nmol DCVG/ml blood
46.1 ± 14.2(2 hr)
13.4 ± 6.6 (4 hr)
pmol DCVG/mg protein
517 ± 250 (3 hr)
138 ± 70 (4 hr)
"Healthy human volunteers (11 males, 10 females) were exposed by inhalation for 4 hr to either 50/60 ppm TCE (3 males, 2 females)
or 100 ppm TCE (8 males, 8 females), and blood was collected at various times both before and after the exposure period. For analysis
of DCVG, blood was deproteinated with acid and acid-soluble extracts were derivatized with iodoacetate and 1-fluoro-2,4-dinitrobenzene for analysis of N-dinitrophenyl-DCVG by ion-exchange HPLC using an amine column, a methanol-acetate solvent system with
gradient elution, and detection at 365 nm. Maximal concentrations of DCVG recovered in whole blood, with the time after exposure
indicated in parentheses, are shown below. Data were reported in (88). Results are the means ± SE of 3 sets of samples for males at
50/60 ppm TCE, means ± SE for 8 sets of samples for males or females at 100 ppm TCE, and the average of 2 sets of samples for
DCVG
)
y-Glutamyltransferase
--
Acivicin
DCVCG
Dipeptidase
|
(3 _
Dipeptidase
DCVC
Cysteine Conjugate
)i-lyase
1,1 0-Phernanthroline
PheGly
-AOAA
0
----
2-Keto aacids
[DCVSH]
females at 50/60 ppm TCE.
dipeptidases to DCVC (90). As will be intracellularly, thus regenerating the cysteine
discussed below, DCVC is the critical conjugate. Hence, administration of
branch point in the metabolism of TCE by NAcDCVC can also produce nephrotoxicity
the GSH conjugation pathway. By this, it is (93,94), and the relative rates of N-acetylation
meant that DCVC can be metabolized furand deacetylation are important in determinther by multiple enzymes to yield both ing the fraction of the mercapturate that can
detoxication products that are excreted and produce toxic metabolites.
reactive species that have been associated
Zhang and Stevens (94) compared the
with nephrotoxicity and are believed to be intracellular disposition and metabolic fate of
associated with nephrocarcinogenicity.
DCVC and NAcDCVC in suspensions of rat
One strategy to validate the function of renal proximal tubules. DCVC was rapidly
the GSH conjugation pathway in bioactiva- transported into the tubules and was rapidly
tion and toxicity has been to use either co- converted to NAcDCVC or was converted to
substrates that are specific for particular steps reactive species that covalently bound to celluto potentiate activity or toxicity or to use spelar proteins. Comparison of the amount of
cific inhibitors of enzymatic steps to inhibit radiolabel covalently bound to cellular propathway flux or toxicity. If the GSH conjuga- teins with 35S-DCVC and 35S-NAcDCVC
tion pathway is responsible for bioactivation showed that a much higher proportion of
and toxicity, then cosubstrates should radiolabel was bound from DCVC than from
enhance toxicity and specific inhibitors NAcDCVC. These data suggested that
should diminish toxicity. As summarized in deacetylation of NAcDCVC is relatively slow.
Figure 5, this strategy has been applied to As described below, a significant portion of
DCVG and its metabolites in both in vivo the DCVG that is formed in the liver is
experiments (85) and in vitro studies with sus- excreted into the bile and is converted there
pensions of isolated rat kidney cells (91). and in the intestines to DCVC. After enteroHence, requirements for hydrolysis of DCVG hepatic circulation, some of this DCVC is
by GGT was demonstrated with L-(aS,5S)-a- converted to NAcDCVC in the liver and then
amino-3-chloro-4,5-dihydro-5-isoxazoleacetic excreted into the plasma and translocated to
acid (acivicin), a potent and irreversible the kidneys. Hence, the relative amounts of
inhibitor of GGT, and with a cosubstrate of DCVC and NAcDCVC that are released
the enzyme. Similarly, inhibitors of dipeptidase from the liver for translocation to the kidneys
activity and of the I-lyase all prevented or will modulate the amount of nephrotoxic and
greatly diminished cytotoxicity in vitro or nephrocarcinogenic metabolites that are
nephrotoxicity in vivo of chemicals that occur ultimately generated within the kidneys.
before the inhibited step.
The NAcDCVC that is secreted into the
Metabolism of DCVC to NAcDCVC. In tubular lumen of the kidneys or that arrives
the classic view of the GSH conjugation path- there by glomerular filtration and is not transway, cysteine conjugates undergo N-acetylaported back into the renal epithelial cell for
tion to yield mercapturates which, because of deacetylation is excreted into the urine.
their polarity, are readily excreted in the Indeed, NAcDCVC has been recovered in the
urine. The N-acetylation reaction is catalyzed urine of rats (54,95,96), mice (97), and
by a cysteine S-conjugate N-acetyltransferase humans (17,96,97) after exposure to TCE.
found in the endoplasmic reticulum (92). This demonstrates that the GSH conjugation
This N-acetyltransferase is distinct from the pathway for TCE occurs in vivo. Urinary
cytosolic enzymes that are polymorphic in NAcDCVC has been considered as a marker
humans (e.g., isoniazid polymorphism). The for flux of TCE through the GSH conjugation
mercapturate can also be deacetylated
pathway, mostly for lack of a better alternative.
188
Nephrotoxicity
Nephrocarcinooenicity
Figure 5. Validation of GSH conjugation and ,B-Iyase pathway
for bioactivation of DCVG. Abbreviations: GlyGly, glycylglycine;
PheGly, phenylalanylglycine; AOAA, aminooxyacetic acid.
As described in the next and subsequent
sections of this review, it is likely that urinary
NAcDCVC represents only a small fraction of
the total flux. Hence, although NAcDCVC
may be reliably used to assess exposure to
TCE, it will not be a very sensitive indicator
owing to the small amounts recovered. More
important for risk assessment, NAcDCVC has
also been specifically considered as a marker of
flux through the pathway that produces the
nephrotoxic and nephrocarcinogenic metabolites of TCE. As described above, NAcDCVC
is a detoxication product, whereas other
metabolites of DCVC are the ones that are
directly associated with renal injury. These
considerations will be discussed further later in
this review in relation to flux through the
oxidative pathway of TCE metabolism and
sex- and species-dependent differences in
metabolism of TCE.
DCVC metabolism by P3-lyase. Early studies
with DCVC described its enzymatic breakdown by a C-S lyase in liver and kidney to
pyruvate, ammonia, and an unidentified sulfurcontaining fragment that formed covalent
adducts with GSH and protein (98). Studies
over the next three decades showed that the
enzymatic activity, the J3-lyase, is a family of
pyridoxal phosphate (PLP)-containing enzymes
that are found in several tissues besides the kidneys, induding rat and human liver (99-103),
intestinal microflora (104,105), and rat brain
(106,107). However, many of these various P3lyase activities are catalyzed by distinct enzymes
with varying substrate specificities or are not
exposed to cysteine conjugates in the normal
pathways of interorgan metabolism.
Although the DCVG that is formed in the
liver and excreted into the bile can be degraded
to
DCVC and should then be available for
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
METABOLISM OF TCE
,B-lyase-mediated catalysis by the intestinal
microflora, these enzymes are not thought to
play a significant role in DCVC metabolism
and no intestinal toxicity is seen with administration of DCVC. As discussed below in the
section on tissue distribution of I-lyase activity, the hepatic P-lyase activity is associated
with kynureninase (100) and is distinct from
the renal I-lyase activities (108), whereas the
renal 13-lyase activity is a catalytic property of
glutamine transaminase K (109-116).
Alberati-Giani et al. (106) reported that rat
liver also expresses the mRNA for the renal
glutamine transaminase K but that the levels of
the mRNA in the liver are much lower than
those in the kidneys. Due to the patterns of
interorgan metabolism and the tissue specificity of enzymes that process GSH conjugates
and related metabolites, hepatic kynureninase
does not appear to play a role in DCVC
metabolism and no liver pathology is observed
after treatment of rats with DCVG or DCVC
(85,86,99). Hence, it is the renal P-lyase activities that are critical for DCVC bioactivation.
The initial 3-lyase activities that were purified from rat (115) and human (113) kidney
cytosol were identified as catalytic activities of
glutamine transaminase K with reported molecular weights of 85-100 kDa. Multiple P-lyase
activities appear to be present in renal cortical
mitochondria, a soluble form present in the
matrix and identified with glutamine transaminase K (116) and a membrane-bound form
that is distinct from glutamine transaminase K
(112). Recent studies from Cooper and colleagues (117,118) have identified an addi-
tional, high-molecular-weight form of renal
[-lyase with apparent molecular weight of 330
kDa. The high-molecular-weight form, like
the low-molecular-weight form, is localized to
the cytosol and mitochondrial matrix but is
immunologically distinct from glutamine
transaminase K and has no similarities to other
known PLP-containing enzymes. Biochemical
properties of the various renal B-lyase activities
are summarized in Table 12. The apparent
regulation of ,-lyase activity by 2-keto acids
(110), particularly in renal mitochondria,
suggests that metabolic state may alter activity
in vivo, thereby influencing toxicity. Keto
acids stimulate renal ,-lyase activity and
potentiate DCVC-induced cytotoxicity in
freshly isolated rat kidney cells because the 1Blyase catalyzes both ,8-elimination and
transamination reactions: During transamination, the PLP cofactor can become "trapped"
in an inactive pyridoxamine phosphate (PMP)
form in the absence of suitable keto acid
acceptors; addition of exogenous keto acids
converts to PLP, thereby restoring activity to
the 3-lyase (110,115).
As illustrated in Figures 1 and 5, DCVC is
metabolized by the 3-lyase to a reactive thiol,
S-(1 ,2-dichlorovinyl)thiol (DCVSH). This
thiol is chemically unstable and rearranges to
reactive species that alkylate cellular nucleophiles (Figure 6). Trapping experiments were
necessary to demonstrate formation of these
metabolites due to their reactive and chemically unstable nature (119). Similar
approaches have been used to trap and identify chemical structures of reactive intermediates from several other haloalkyl and
haloalkenyl cysteine S-conjugates (119-121).
DCVC metabolites generated by the
catalytic action of the 1-lyase form covalent
adducts with cellular nucleophiles, including
proteins. Bull and colleagues (122,123) suggested that measurement of the acid-labile
adducts could be used as an index of flux
through the ,B-lyase pathway. While this
would seem to make sense, as it is the quantity of the reactive species that is formed that
can produce cellular injury and not, as discussed below, the quantity of stable endproducts that are excreted into the urine (e.g.,
mercapturates), the methodology is potentially complicated by artifacts due to chemical
instability of the molecules being quantified.
Additionally, Eyre et al. (122,123) found
that apparent net activation of TCE by the
1-lyase pathway and apparent acid-labile
adduct formation were greater in mice than
in rats, which does not correspond to the
higher susceptibility of rats to renal toxicity
and carcinogenesis, although adduct formation did correspond to increased cell replication rates. Hence, the relationship between
adduct formation and effects on cellular
function and homeostasis are very complex
and require further study.
MacFarlane et al. (124) showed that pretreatment of rats with a single, subtoxic dose
of the mercapturate of a nephrotoxic halogenated hydrocarbon produced a 1.5- to 3fold increase in renal 1-lyase activity and an
increase in the cognate mRNA. These results
suggest that prior or continuous exposures to
low, subtoxic doses of certain cysteine or Nacetylcysteine conjugates may enhance the
capability to generate reactive species by the
P-lyase after subsequent exposures to toxic
doses of these conjugates or their precursors.
Although little is currently known about the
genetic regulation of the 1-lyase, these data
suggest that differential expression of the 13lyase may need to be taken into account in a
risk assessment for chemicals whose nephrotoxicity is mediated by this enzyme.
Direct demonstration of the function of
the 1-lyase in vivo in either experimental animals or humans has not been possible with
DCVC because of the reactive nature of the
metabolite. However, Iyer et al. (125) recently
reported the identification of a metabolite of
compound A (2-[fluoromethoxy]-1 , 1,3,3,3pentafluoro-1-propene) that could only have
arisen by ,B-lyase-dependent metabolism.
Hence, this is the first study to directly show
function of the 1-lyase in vivo in humans.
DCVC metabolism by other enzymes.
Although the 1-lyase is considered to be the
major bioactivation enzyme for DCVC, other
bioactivation enzyme activities have been
described, and some of these may have relevance to risk assessment. One of these other
enzymes is the renal L-a-hydroxy (L-amino)
acid oxidase (HAO), which can catalyze formation of the keto acid analogue of DCVC
through an iminium ion intermediate, and
this then decomposes to release DCVSH
(126,127). This route of metabolism for
DCVC has been estimated to account for as
much as 35% of bioactivation in male F344
rats. HAO, however, is present only in rat
kidney and is absent from human kidney.
Thus, this pathway is irrelevant for human
health risk assessment.
Table 12. Biochemical properties of mammalian renal cysteine conjugate P-lyase activities.
Description
Property
Cytosol and mitochondria (matrix and outer membrane)
Subcellular localization
Predominantly S3 segment of proximal tubule; also in S, and S2
Intrarenal localization
Low-molecular-weight form: homodimer of 45-50 kDa each; aggregate M,
Molecular weight and
reported of 85-100 kDa; identical to glutamine transaminase K
subunit composition
High-molecular-weight form: 330 kDa; at least two subunits of M,70 and 50
kDa; distinct from glutamine transaminase K
Pyridoxal phosphate
Cofactor
Both forms: good leaving group on beta carbon; haloalkyl or haloalkenyl
Substrate specificity
cysteine S-conjugates are substrates.
Low-molecular-weight form: nonhalogenated, alkyl-substituted conjugates are
not substrates.
High-molecular-weight form: leukotriene E4 as a substrate.
Both forms: 2-keto acids with relatively hydrophobic substituents on beta
Cosubstrate specificity
carbon (e.g., 2-keto-4-methiolbutyrate, phenylpyruvate).
High-molecular-weight form: also activated by polar 2-keto acids (e.g.,
2-ketoglutarate and pyruvate).
or transamination followed by reverse Michael elimination
Beta-elimination
Reaction mechanism
acid
Aminooxyacetic
Inhibitor
VrnviTonmental Health Perspectives * Vol 108, Supplement 2 * May 2000
189
LASH ET AL
Cl
~ ~3
\COS
H~
\/
Cl
1, DCVC
coo-
I
cls
2, DCVSH
HNEt.,
H,0
4b
Nu
Cl'
Figure 6. Scheme of formation of reactive metabolites from DCVC. Abbreviations: HNEt2, diethylamine; Nu, cellular
nucleophile. Metabolites: 1, DCVC; 2, DCVSH; 3a, chlorothioketene; 3b, chlorothionoacetyl chloride; 4a, N,Ndiethylchlorothioacetamide; 4b, covalent adduct with cellular nucleophile; 5a, chlorothionacetic acid; 5b, CAA.
Metabolite 4a is formed by trapping reactive metabolites 3a/3b with HNEt2. Metabolites 5a and 5b are stable end
products that are formed by hydrolysis. Modified from Dekant et al. (119).
Sulfoxides are known metabolites of
cysteine conjugates that can be recovered in
the urine as stable end products. Elfarra and
colleagues (128,129) identified and subsequently purified an enzymatic activity from rat
liver and kidney microsomes that metabolizes
DCVC to its sulfoxide. This enzyme was subsequently shown to be a flavin-containing
monooxygenase (FMO), initially thought to
be related to FMOIA1 (129), but more recent
data suggest that FMO3 is the isoform responsible for DCVC oxidation (130). Unlike other
cysteine conjugate sulfoxides, DCVC sulfoxide
is unstable and reacts with cellular nucleophiles, including GSH (131). The potential
significance of this activity for overall TCE or
DCVC bioactivation and for human health
risk assessment is undear, but DCVC sulfoxide was shown to be markedly more nephrotoxic to rats and more cytotoxic in freshly
isolated rat renal cells than DCVC (132). This
finding warrants further investigation.
Werner et al. (133) recently showed that
sulfoxides can arise by a different pathway. In
their studies, they found that NAcDCVC and
190
the mercapturate of perchloroethylene (PER)
metabolized by rat liver microsomes to
the corresponding sulfoxides by CYP3A and
not by an FMO. These investigators also
found that the N-acetylcysteine conjugate sulfoxides were markedly more cytotoxic in
freshly isolated rat renal cortical cells than the
corresponding mercapturates, similar to the
situation with cysteine conjugate sulfoxides.
This report adds an additional dimension to
known bioactivation mechanisms of DCVC.
Due to the efficient processing of mercapturates by the liver, which leads to their delivery to the kidneys, and the much lower
activity of CYP3A in the kidneys as compared
with the liver, this pathway is not likely to be
quantitatively important overall or even in
comparison to the P-lyase pathway.
An additional although minor fate of the
reactive DCVSH generated by the ,-lyase and
other enzymes is methylation, which occurs by
a thiomethyltransferase in the intestinal
microflora (134). The thiomethyltransferase
uses S-adenosylmethionine to transfer a methyl
group to the thiol, forming the methylthio
were
derivative, which is subsequently oxidized to
the methylsulfinyl (CH3SO-) and methylsulfonyl (CH3SO2-) analogs. The methylthio
derivatives are nonpolar and are generally
excreted into the feces. Quantitatively, this
pathway is probably minor for TCE, accounting for no more than 0.05-0.1% of the total
excreted metabolites of TCE.
Membrane transport of GSH-derived
metabolites of TCE. Membrane transport
processes play an important role in the tissue
distribution of the various GSH conjugates
and related metabolites [(89); also see discussion below on interorgan metabolism). There
are three GSH-derived metabolites of TCE
whose membrane transport is important for
understanding tissue distribution: DCVG,
DCVC, and NAcDCVC. For DCVG, both
efflux from the hepatocyte and membrane
transport processes in renal proximal tubular
cells are toxicologically important. For both
DCVC and NAcDCVC, uptake into renal
proximal tubular cells is the relevant process.
As discussed above, there are two primary
sources or sites of formation of DCVG-the
liver and the kidneys. For DCVG that is generated by renal GSTs, further processing
occurs by efflux of intracellular DCVG by
transport across the brush-border membrane
into the lumen, where it can then be metabolized by GGT and dipeptidases to generate
DCVC. Efflux of GSH and GSH S-conjugates
into the lumen by transport across the brushborder membrane is viewed as an essential step
in the turnover of these compounds because
the active site of GGT is extracellular (135). In
fact, inhibition of GGT in vivo produces
marked glutathionuria in mice (135).
Presumably, a marked increase in urinary
excretion of GSH conjugates would also occur
under similar conditions. Although this transport step has not been specifically quantitated
for DCVG, Inoue and Morino (136) determined the mechanism and kinetics of trans-
for GSH in renal brush-border
membrane vesicles. GSH uptake was dependent on the membrane potential and exhibited a Km for GSH of 0.21 mM and a V,, of
1.15 nmol/min per mg protein. It can be
expected that brush-border membrane DCVG
transport will likely exhibit similar kinetics.
For DCVG that is generated in the liver,
there are two additional steps, efflux across the
canalicular membrane into the bile or across
the sinusoidal membrane into the plasma, and
extraction by the kidneys. Hepatic GSH conjugates are transported primarily into bile by
the canalicular multispecific organic anion
transporter (cMOAT), which is an ATPase
that mediates efflux of various GSH conjugates and organic anions (137-140). Again,
transport of DCVG by this carrier has not
been specifically studied. However, kinetics
for transport of the model GSH conjugate
port
Environmental Health Perspectives
*
Vol 108, Supplement 2
*
May 2000
METABOLISM OF TCE
S-(2,4-dinitrophenyl)glutathione (DNPSG)
have been characterized. Km (pM DNPSG)
and Vm,, (nmol translocated/min per mg protein) values of 71 and 0.34, respectively, were
reported by Akerboom et al. (137), whereas
Niinuma et al. (140) reported values of 18
and 1.15, respectively. Comparisons with
other model GSH conjugates reveal some
variability in rates of transport (up to a 5-fold
range), suggesting that these values for
DNPSG should only be used as a rough estimate for the transport of DCVG from the
hepatocyte into the bile. Although data in
normal human liver are not available, Olive
and Board (141) characterized efflux of
DNPSG from eight different cultured human
liver cell lines, including Jurkat, HL-60,
HepG2, and HeLa cells. A 10-fold range of
transport rates was observed. These cells also
exhibited a 6-fold range of intracellular GSH
contents and a 5-fold range of GST activities.
However, there was no correlation between
transport activity and either GSH content or
GST activity. Hence, although transport data
from normal human liver is unavailable, these
studies in cell lines suggest that considerable
variations in transport capacity may exist.
Besides the luminal transport of DCVG,
plasma DCVG that does not undergo
glomerular filtration may gain access to the
renal proximal tubular cell by transport across
the basolateral plasma membrane (142). This
transport occurs by the Na+-coupled system
described for GSH (143,144). Transport of
both GSH and GSH S-conjugates is inhibited by probenecid and p-aminohippurate,
suggesting that the organic anion carrier may
be involved in the transport. Another study
on renal plasma membrane GSH transport
(145) indicates the additional involvement of
plasma membrane dicarboxylate carriers.
Rates of DCVG transport into membrane
vesicles were extremely rapid (22 nmol/min/
per mg protein with 5 mM DCVG) and
transport was inhibited by GSH and
y-glutamyl amino acids, but not by DCVC
(142). Once inside the renal proximal tubular cell, DCVG will be translocated to the
brush-border membrane for efflux into the
lumen and metabolism as described above.
Once DCVG is secreted into the bile, a
significant portion of it will be metabolized
by either biliary or intestinal GGT and
dipeptidases to form DCVC. The transport
processes relevant for DCVC include uptake
into the hepatocytes (enterohepatic circulation) and uptake into the renal proximal
tubular cell. Again, little data are available on
rates of DCVC transport into the liver, with
the exception of one in vitro study. Simmons
et al. (146) characterized DCVC uptake by
isolated canalicular membrane vesicles from
rat liver. DCVC uptake was Na+ independent
and exhibited Km and Vm,, values of 155 ,uM
and 4.7 nmol/min per mg protein, respectively.
This step thus appears to have a greater capacity than any of the steps for DCVG transport.
Renal extraction of plasma DCVC can
occur by either glomerular filtration and
uptake across the brush-border membrane or
by uptake across the basolateral membrane.
Transport across the brush-border membrane
from rat kidney cortex was reported to be Na+
dependent and exhibited Km and V, values
of 225 pM and 3.1 nmol/min per mg protein,
respectively (147). In contrast, Wright et al.
(148) found that DCVC uptake in brush-border membrane vesides from rabbit renal cortex
was Na+-independent and exhibited Km and
V, values of 0.45 mM and 60 nmol/min per
mg protein. Brush-border membrane transport
of DCVC was unaffected by NAcDCVC or
several other organic anions but appears to
occur by the same carrier that transports neutral amino acids such as phenylalanine. In contrast to this, basolateral transport of DCVC
occurs by both Na+-dependent and Na+-independent mechanisms and the Na+-dependent
process interacts with the transport of
NAcDCVC and organic anions (149,150).
Rates of DCVC uptake by isolated renal proximal tubular cells, which reflect primarily transport processes occurring at the basolateral
membrane, were approximately 2-fold higher
for DCVC than for DCVG (142,150). Thus,
as with canalicular membrane transport, renal
transport of DCVC appears to be significantly
faster than that of DCVG.
After release from the hepatocyte, DCVG
will undergo interorgan translocation and/or
metabolism. In its translocation to the kidneys, a portion of it will first be returned to
the liver before efflux into plasma (renalhepatic circulation). Some of the DCVG will
be metabolized extrahepatically to DCVC
and some of this will be N-acetylated, either
extrahepatically or in the liver to form
NAcDCVC. Hence, the mercapturate is also
a form to which the kidneys are exposed.
Transport of NAcDCVC into renal proximal
tubular cells occurs readily and presumably
by the organic anion carrier (93,94). Kinetic
parameters for transport at specific membrane
sites are unavailable, but estimates appear to
be similar to those for DCVC. Transport of
NAcDCVC is not believed to be limiting for
nephrotoxicity, as described above. Rather,
the intracellular deacetylation reaction likely
determines the extent of regeneration of
DCVC and the degree of cytotoxicity.
Tissue Distribution and Sex- and
Species-Dependent Heterogeneity
of GSH-Dependent Metabolism of TCE
The selective tissue distribution of the various
enzymes of the GSH-dependent metabolism
of TCE plays a critical role in determining
the target organ specificity of DCVC and the
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
reactive species that are derived from its
further metabolism. Hence, a precise knowledge of the relative activities of each of the
major enzymes of the pathway is important,
to help both in understanding how toxicity
occurs and in assessing the relevance of the
GSH conjugation pathway relative to the
oxidative pathway in TCE-induced toxicity.
The liver and kidneys are the major sites of
GSH-dependent metabolism of TCE,
although biliary and intestinal metabolism
also play significant roles.
Although most cell types express some
GST activity, the liver is by far the predominant site of GSH conjugation. Table 13 summarizes measurements of GST activity with
1-chloro-2,4-dinitrobenzene as substrate in
cytosolic fractions from male and female F344
rat and B6C3F, mouse liver and kidney. With
the exception of the female rat, specific activities (normalized to protein) are slightly to
modestly lower in kidney cytosol than in
those from liver cytosol. However, total activity in the liver will be much higher than in the
kidneys due to the larger tissue size.
For chemicals such as TCE that undergo
GSH conjugation and eventually are translocated to the kidneys for further metabolism,
it has generally been assumed that most if not
all of the GSH conjugation occurs in the
liver. However, Lash et al. (151) showed that
TCE is conjugated with GSH in freshly isolated kidney cells from male F344 rats,
although at rates that are only 5 to 10% of
those in isolated hepatocytes (Figure 7). Koob
and Dekant (152) also demonstrated that
hexafluoropropene, a nephrotoxic fluoroalkene, can undergo GSH conjugation in
the kidney as well as in the liver. Thus, the
entire GSH conjugation pathway can occur
within the target tissue. Although the majority of the flux involves interorgan processes
(see below), with the liver catalyzing the
majority of the initial step in the pathway,
this intraorgan process should be taken into
account in refinements of PBPK models and
risk assessments for TCE.
A summary of measurements of DCVG
formation from TCE in liver and kidney
cytosol and microsomes from male and
female F344 rats and B6C3F, mice
(153,154) is given in Table 14. The results
show some dramatic sex- and species-dependent differences in GSH conjugation of TCE.
Although overall GSH conjugation in male
rats is clearly higher than that in female rats,
no detectable DCVG formation was observed
in male rat liver microsomes, whereas DCVG
formation in female rat liver microsomes
occurred at approximately twice the rate as in
the cytosol. Rates of metabolism in mice in
both kidney and liver fractions were also generally faster in males than in females and were
markedly higher than corresponding rates in
191
LASH ET AL.
rats. This last point brings up an interesting
correlation with the covalent adduct formation reported by Bull and colleagues (93,94).
As summarized above, 2-fold higher rates of
covalent adduct formation from 35S-labeled
DCVC were found in mouse kidney compared to those in rat kidney. Hence, both
higher rates of adduct formation and GSH
conjugation of TCE occur in the mouse kidney, although the rat is the species in whom
renal tumors are observed.
Kinetics of GSH conjugation of TCE in
subcellular fractions from pooled human
liver and kidney samples are summarized in
Table 15. Although analysis could only
detect a single, kinetically distinct process for
DCVG formation in kidney fractions, GSH
conjugation of TCE in both liver cytosol and
Table 13. Measurements of GST activity in liver and
kidney cytosol from male and female F344 rats and
B6C3F, mice.a
Activity
Sex
Tissue
Species
(mU/mg protein)
Male
Mouse
Liver cytosol
0.78 ± 0.28
Male
Mouse
Kidney cytosol 0.70 ± 0.09
Female Mouse
Liver cytosol
0.49 ± 0.03
Female Mouse
Kidney cytosol 0.34 ± 0.04
Rat
1.10 ± 0.44
Male
Liver cytosol
Male
Rat
Kidney cytosol 0.55 ± 0.10
Female Rat
Liver cytosol
0.28 ± 0.06
Female Rat
Kidney cytosol 0.34 ± 0.08
'GST activity was measured spectrophotometrically with 1 mM
1-chloro-2,4-dinitrobenzene as substrate by determining formation of 2,4-dinitrophenylglutathione at 340 nm. Results are the
means ± SE of measurements from 6 experiments for rats and 4
experiments for mice. Data reported in ( 153).
15-
-(
10-
I:I Hepatocytes
o1 Kidney Cells
0
2 mM TCE
1 mM TCE
Figure 7. GSH conjugation of TCE in suspensions of
freshly isolated hepatocytes and renal cortical cells from
male F344 rats. Isolated hepatocytes (2 x 106 cells/ml)
or renal cortical cells (8 x 106 cells/mL) were incubated
with 1 or 2 mM TCE in the presence of 5 mM GSH at
30°C for 60 min. DCVG formation was measured by ionexchange, reversed-phase high-performance liquid chromatography after derivatization with iodoacetate and
1-fluoro-2,4-dinitrobenzene, and was detected by
absorbance at 365 nm. Results are the means ± SE of 3
experiments. See (1501 for other details.
192
microsomes were resolved into two kinetically
distinct processes. Maximal rates of DCVG
formation in human liver and kidney were
only modestly lower than those in rat liver
and kidney but were approximately 5-fold
lower than those in the mouse. Hence, it
appears that any limitation in the generation
of reactive metabolites of TCE from this
pathway does not exist at the initial step
where DCVG formation occurs. Rates of
GSH conjugation of TCE in human liver
and kidney subcellular fractions reported
here are up to an order of magnitude greater
than those reported by Green et al. (154). In
incubations with 1.9 mM TCE and 5 mM
GSH, Green and colleagues found rates of
DCVG formation of 2.5, 1.6, and 0.19
pmol/min per mg protein in liver cytosols of
male mice, rats, and humans, respectively. In
contrast, in incubations with 2 mM TCE
and 5 mM GSH, Lash and colleagues
(151,153,155) reported rates of DCVG formation of 408, 122, and 5,770 pmol/min
per mg protein in liver cytosols of male mice,
rats, and humans, respectively. Similarly,
Green et al. (154) were unable to detect net,
enzymatic formation of DCVG in incubations with rat kidney subcellular fractions. In
contrast, Lash and colleagues (151,153)
reported a rate of DCVG formation in incubations of male rat kidney cytosol with 2
mM TCE and 5 mM GSH of 7.5 pmol/min
per mg protein and a rate of 0.7 nmol/min
per mg protein in pooled human kidney
cytosol (154).
The controversy between these results has
not been resolved. Differences in analytical
methods (radiolabeled substrate with high performance liquid chromatography (HPLC) separation versus derivatization and HPLC
Table 14. Summary of metabolism of TCE by GSH conjugation in kidney and liver subcellular fractions from
male and female F344 rats and B6C3F, mice.a
DCVG formation
(nmol/mg protein or
106 cells per 60 min)
Subcellular fractions
Male
Female
Rat kidney cells
0.48±0.02 0.65±0.15
Rat hepatocytes
9.70 ± 0.29 2.67 ± 0.69
Rat kidney cytosol
0.45 ± 0.22 0.32 ± 0.02
Rat kidney microsomes
ND
0.61 ± 0.06
Rat liver cytosol
7.30 ± 2.80 4.86 ± 0.14
Rat liver microsomes
10.3 ± 2.8 7.24 ± 0.24
Mouse kidney cytosol
5.60 ± 0.24 3.70 ± 0.48
Mouse kidney microsomes 5.47 ± 1.41 16.7 ± 4.7
Mouse liver cytosol
24.5±2.4 21.7±0.9
Mouse liver microsomes
40.0±3.1
25.6±0.8
ND, not detectable; limit of detection was 0.05 nmol/mg protein
106 cells. &Results are means ± SE of measurements from 3
separate cell or tissue preparations and are for 2 mM TCE incubated with 5 mM GSH for 60 min. DCVG formation was measured after derivatization of acid extracts with iodoacetate and
1-fluoro-2,4-dinitrobenzene, separation by ion-exchange, gradient HPLC on an amine column using a methanol-acetate mobile
phase and detection of N-dinitrophenyl DCVG at 365 nm. Data
reported in ( 151,153).
or
separation) may contribute to the discrepancies
in measured rates. Beyond this, no other
explanations are available to explain the discrepancies. Green and colleagues (154) also
reported that formation of DCVG was linear
with respect to incubation time for at least 60
min, whereas Lash et al. (151,153) showed
the expected saturation kinetics and maximization versus incubation time of an
enzyme-catalyzed process.
Measurements of GGT activity in liver
and kidney subcellular fractions from male
and female rats and mice and pooled human
liver and kidney samples show clearly that at
least in rodents, insignificant GGT activity is
present in the liver (Table 16). Human liver
exhibits relatively low activity of GGT
(approximately 3% of that in the kidneys).
Only modest sex-dependent differences were
observed in GGT activity. Rats exhibited the
highest activity of GGT, with humans
exhibiting approximately 50% of that in rats
and mice exhibiting only 20% of that in rats.
Higher GGT activity provides more substrate
for the P-lyase reaction, which would generate higher amounts of reactive species. Hence,
these differences may contribute to speciesdependent differences in overall flux of TCE
through the GSH conjugation pathway.
Hinchman and Ballatori (156) compared renal and hepatic GGT activity in several mammalian species. Although the
kidneys contain the highest levels of GGT
activity in all species examined, including
humans, specific activities (i.e., mU/mg protein) can vary up to 10-fold or more
between species. Furthermore, although
adult liver contains relatively low levels of
GGT activity, specific activities can also vary
up to 10-fold or more. Hence, while the
ratio of GGT specific activity in kidney to
that in liver was reported as 875 in rats and
413 in mice, it was only 100 in rabbits and
19 in pigs. Humans exhibit tissue distributions similar to rabbits. Use of rats to model
Table 15. Kinetics of GSH conjugation of TCE in human
kidney and liver subcellular fractions.a
Vmax(nmol DCVG/ Km
Tissue/subcellular fraction min/mg protein) (pM TCE)
Kidney
Cytosol
0.81
26.3
Microsomes
6.29
167
liver
Cytosol: low affinity
8.77
333
Cytosol: high affinity
4.27
22.7
Microsomes: low affinity
3.10
250
Microsomes: high affinity
1.42
29.4
'Activity of GSH conjugation of TCE was measured in cytosol
and microsomes from pooled human liver and kidney samples.
Fractions (1 mg protein/ml) were incubated with 7.8 pM-1 mM
TCE and 5 mM GSH at 37T; DCVG formation was quantitated
by HPLC analysis after derivatization. Kinetic parameters were
calculated from Eadie-Hofstee plots and are derived from measurements from 3 separate incubations. Results were reported
in ( 155).
Environmental Health Perspectives * Vol 108, Supplement 2 ' May 2000
METABOLISM OF TCE
interorgan GSH and GSH S-conjugate
metabolism thus overestimates the role of
these pathways in turnover. This is not to
say, however, that the interorgan pathways
do not play a significant role even in
humans, since total renal activity is still sev-
eralfold higher than that of total hepatic
activity.
A scheme summarizing the interorgan
processes involved in GSH conjugation and
subsequent metabolism of TCE is shown in
Figure 8. Processes occurring in the liver,
small intestine, kidneys, bile, and plasma are
highlighted. Although most studies that were
performed in the development of this model
have used the rat, the interorgan metabolism
model is valid in humans, although fluxes and
distributions of metabolites in the various
compartments (i.e., plasma, bile) will vary.
TCE is shown as having two fates: being
taken up by the liver where it is conjugated
with GSH or being taken up by the kidney
where it may also be conjugated with GSH.
The two principal interorgan pathways are the
enterohepatic and renal-hepatic circulations.
The determining factors in these pathways are
the following: a) high activity of GST in the
liver; b) efficient transport systems in liver cells
for efflux of GSH conjugates into bile or
plasma; c) relatively high activity of GGT on
renal brush-border membranes and relatively
low activity of GGT in liver; and d) selective
presence of a transport system for uptake of
GSH conjugates on renal basolateral
membranes and the absence of an uptake system in liver cell plasma membranes.
GSH conjugation of TCE was demonstrated in isolated human hepatocytes
(Figure 9). Rates of DCVG formation,
although lower than those in rat hepatocytes
by approximately 2- to 3-fold, were not so
low or difficult to detect that one would conclude that flux through the GSH conjugation
pathway is too low to account for a measurable and possibly significant portion of total
TCE metabolism. Of course, this is only the
initial step in the pathway with subsequent
branches leading to excretion of nontoxic
products in addition to the branches that
produce toxic metabolites. Hence, it is critical to quantitate formation of the metabolites
that directly produce cellular injury.
As emphasized above, the key step in the
bioactivation of TCE by the GSH conjugation pathway is the ,-lyase step. Reports of
,-lyase activity in the literature show a fair
of variability, depending in part on
the cysteine conjugate substrate used, the
assay method used, and even the laboratory
reporting the data. Table 17 summarizes
activity data or, when available, kinetic parameters, for ,B-lyase activity in kidney cytosol
or homogenates from rats, mice, and
humans. Km values for cysteine conjugates
are, with the exception of two reports, > 1
mM, indicating that the 1-lyase is a relatively low-affinity pathway. Activities vary
considerably among substrates. In the one
study (157) where 3-lyase activity in kidney
cytosol from male and female rats, mice and
humans was measured together; the results
showed that Km values for S-(1,2,2trichlorovinyl)-L-cysteine (TCVC) were
markedly higher and Vmax values were
markedly lower for mice and humans as
compared to rats. This species difference is
consistent with the higher susceptibility of
amount
TCE
LIVER
m
TCE
BILE
DCVG
DCVC
-*
PLASMA
NAcDCVC
AF
4-
Table 16. Measurements of GGT activity in liver and
kidney from male and female F344 rats and B6C3F, mice
and pooled human liver and kidney samples.a
Activity
Fraction
Sex
Species Tissue
(mU/mg)
0.07 0.04
Male Mouse Liver
Cytosol
Microsomes 0.05 0.04
1.63 0.85
Kidney Cytosol
Microsomes 92.6 15.6
0.10 0.10
Female Mouse Liver
Cytosol
Microsomes 0.03 0.03
0.79 0.79
Kidney Cytosol
Microsomes 69.3 14.0
< 0.02
Liver
Male Rat
Cytosol
< 0.02
Microsomes
< 0.02
Kidney Cytosol
Microsomes 1,570 100
< 0.02
Liver
Female Rat
Cytosol
< 0.02
Microsomes
< 0.02
Kidney Cytosol
Microsomes 1,840 40
8.89 3.58
Male Human Liver
Cytosol
29
Microsomes
13.2 1.0
Kidney Cytosol
Microsomes 960 77
NAcDCVC DCVG
SMALL
INTESTINE
±
±
GLOMERULAR
FILTRATION
KIDNEY
±
TCEl
±
1
±
±
_
±
±
DCVG
DCVC-
* DCVG
Nephrotoxicity
Nephrocarcinogenicity
*
DCVG
+
DCVC
DCVSH
±
DCVC
DCVC
±
±
NAcDCVC
-
1A
±
±
_ *NAcDCVC
'GGT activity was measured spectrophotometrically with 1 mM
y-glutamyl-p-nitroanilide and glycylglycine as substrates by
determining formation of p-nitroanilide at 410 nm. Results are
the means SE of measurements from 6 experiments for rats, 4
experiments for mice, and 3-9 measurements from humans,
PLASMA
±
except for human kidney microsomes, which is the average of 2
measurements. Data reported in (153,155).
Environmental Health Perspectives
*
Vol 108,
NAcDCVC
417
URINE
Figure 8. Scheme for interorgan processing and metabolism of TCE by the GSH conjugation pathway.
Supplement
2
*
May
2000
193
LASH ETAL.
0.25
'
0.20
i0.15
0.10
n
0.05
0.00
0
2500
5000
7500
TCE (ppm)
10000
Figure 9. GSH conjugation of TCE in isolated human
hepatocytes. Isolated hepatocytes (2.0 x 106 cells/mL)
were isolated from livers of human donors and were
incubated in sealed flasks with the indicated concentration of TCE in the headspace for 120 min. DCVG formation was assayed in perchloric acid extracts of aliquots
of cells by ion-exchange, reversed-phase high-performance liquid chromatography after derivatization with
iodoacetate and 1-fluoro-2,4-dinitrobenzene. Derivatives
were detected spectrophotometrically at 365 nm and
were quantified with respect to authentic standards.
Data reported in (155).
rats to kidney toxicity and cancer than other
species. Km and Vmax values for male rats
were also lower and higher, respectively,
than those for female rats, which is consistent
with male rats being more susceptible than
female rats. The central questions for risk
assessment, therefore, are: a) Can studies in
male rats be extrapolated to humans? and b)
Is P-lyase activity in human kidney high
enough to generate sufficient amounts of
reactive metabolites to produce nephrotoxicity and kidney cancer? Additional studies are
clearly necessary to accurately determine flux
through this pathway.
Elfarra and Hwang (158) used the model
cysteine conjugate S-(2-benzothiazolyl)-L-cysteine (BTC) as an in vivo marker of flux
through the 3-lyase pathway. BTC was originally synthesized as a convenient assay substrate for the P-lyase (99) because it is
converted to a stable thiol metabolite, 2-mercaptobenzothiazole (MBT), which can be
readily measured spectrophotometrically.
They administered BTC to male and female
rats and male mice, rats, hamsters, and guinea
pigs. BTC is metabolized by the P-lyase and is
recovered in the urine as either MBT or MBT
glucuronide, both of which are chemically
stable and readily measured. Male rats released
more metabolite into the urine than female
rats and of the four species compared; mice,
hamster, and rats excreted comparable levels
of total metabolites, whereas the guinea pig
excreted more than twice the amount of
metabolites as the other species. For the
guinea pig, however, the higher excretion rate
was not attributable to higher P-lyase activity.
Rather, the guinea pig is unique in having
extremely low N-acetylation activity and
extremely high deacetylation activity (159),
which leads to more BTC being available for
P-lyase-mediated metabolism. Hence, measurements of MBT plus MBT glucuronide
plus the mercapturate of MBT may provide a
useful means of accurately determining flux
through the ,B-lyase pathway.
The importance of N-acetylation versus
N-deacetylation in determining cysteine
conjugate nephrotoxicity was highlighted in
a recent study by Lau et al. (159), in which
they determined rates of the forward and
reverse reactions for mercapturate formation
from S-(2,5-dihydroxyphenyl)-L-cysteine,
which is a cysteine conjugate derived from
Table 17. Compilation of selected data on cysteine conjugate f-lyase activity from rat, mouse, and human kidney cytosol or homogenates reported in the literature.
Specific activity
Vmax
Reference
Km(mM)
6.2 ± 1.0
BTC (2 mM)
(99)
3.1 ± 0.2
DCVC (1 mM)
(108)
0.9±0.1
BTC(1 mM)
M F344 rat
BTC (0.5 mM)
20.5
Kidney cytosol
(86)
M SD rat
DCVC (1 mM; + 0.1 mM KMB)
0.02
Kidney cytosol
(115)
M F344 rat
CTFC (4 mM)
22.5 ± 2.5
Kidney cytosol
(166)
M F344 rat
BTC
Kidney cytosol
74.8 ± 6.5
1.66 ± 0.19
(112)
CTFC
11.6 ± 1.6
1.78 ± 0.17
DCVC
38.3 ± 1.4
1.36 ± 0.05
±
M SD rat
DCVC (1 mM; + 0.5 mM KMB)
3.05 0.27
Kidney homogenate
(111)
M Wistar rat
DCVC (5 mM)
1.24
Kidney cytosol
(167)
TFEC (5 mM)
2.64
BTC (1 mM) (+ 0.1 mM KMB)
0.70
M F344 rat
2.2
Kidney cytosol (purified enzyme)
DCVC
0.26
(127)
Kidney homogenate
DCVC (1 mM)
4.0 ± 1.0
PCBC (1 mM)
4.5 ± 0.3
M F344 rat
TCVC
Kidney cytosol
4.00 ± 0.11
0.68 ± 0.06
(157)
F F344 rat
TCVC
Kidney cytosol
3.64 ± 0.41
1.26 ± 0.21
(157)
M B6C3F1 mouse
Kidney cytosol
TCVC
1.15 ± 0.31
5.69 ± 2.22
(157)
F B6C3F1 mouse
Kidney cytosol
TCVC
1.66 ± 0.27
4.43 ± 1.42
(157)
M human
Kidney cytosol
TCVC
0.49 ± 0.07
2.53 ± 0.09
(157)
F human
Kidney cytosol
TCVC
0.64 ± 0.54
2.67 ± 2.11
(157)
M human
Kidney cytosol
BTC (2 mM)
0.7
(113)
M F344 rat
Kidney cytosol
BTC (2 mM)
6.5
M Wistar rat
Kidney cytosol
TCVC (0.3 mM)
5.2 ± 0.7
(168)
M Wistar rat
Kidney cytosol
DCVC (5 mM; + keto acids)
2.56
(118)
Abbreviations: CTFC, S-(2-chloro-1,1,2-trifluoroethyll-i-cysteine; F, female; M, male; PCBC, S-(1,2,3,4,4-pentachlorobutadienyl)-L-cysteine; TFEC, S-(1,1,2,2-trifluoroethyl)-L-cysteine. Note: Where means ±
SE values are shown, data are from 3-7 separate experiments. Where single values are shown, they are from steps during enzyme purification. 1 mU = nmol product formed/min. BTC is a synthetic cys-
Species
M SD rat
M SD rat
Tissue
Substrate
(nU/mg protein)
(mU/mg protein)
Kidney homogenate
Kidney cytosol
teine conjugate that was developed to assay the ,-lyase because it forms a chemically stable thiol metabolite, 2-mercaptobenzothiazole, which can readily be measured spectrophotometrically 199).
194
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
METABOLISM OF TCE
GSH conjugation of 1,4-benzoquinone, and
subsequent metabolism, in several mammalian species. The ratios of N-deacetylase
to N-acetylase activity in these species
showed striking differences: F344 and
Sprague-Dawley rats exhibited ratios of 0.03
and 0.02, respectively; golden Syrian hamster exhibited a ratio of 0.04, which is similar to that in rats; two mouse strains
(BALB/c and B6C3F1) exhibited modestly
higher ratios of 0.16 and 0.14, respectively;
in contrast to the rat and mouse strains,
guinea pigs exhibited a ratio of 4.57.
Susceptibility to renal injury at higher doses
of the quinone correlated with the ratios in
that guinea pigs were the most and rats the
least susceptible. At lower doses, however,
rats were the most susceptible due to their
exceptionally high GGT activity.
Birner et al. (97) also assessed deacetylation
activity in cytosol from human, Wistar and
F344 rat and NMRI mouse kidney using
NAcDCVC as the substrate. The rates of
deacetylation varied by less than 3-fold across
species, varying from 0.35 nmol DCVC
formed/min per mg protein in the Wistar rat
to values of 0.41, 0.61, and 0.94 in human,
F344 rat, and NMRI mouse. They concluded
that species-dependent differences in deacetylation activity will influence the amounts of
mercapturates recovered in urine.
Varations among Humans
There is much more knowledge about genetic
polymorphisms and variations in P450 reactions in humans than there is for GSTs.
However, increasing attention is being placed
on these potential differences and their toxicological consequences. Analysis of rates of
GSH conjugation of TCE in cytosol and
microsomes from 20 individual human liver
donors has revealed that considerable variation exists (76). Overall, rates of GSH conjugation varied 3.4-fold in liver cytosol,
exhibiting a range of rates of 28.3-94.9 nmol
DCVG formed/20 min per mg protein. The
donor pool comprised 9 males and 11
females. There were no significant sex-dependent differences. The liver microsome donors,
which comprised 5 males and 15 females,
exhibited an overall variation in activity of
8.5-fold, with a range of values from 8.06 to
69.2 nmol DCVG formed per 20 min per
mg protein. As with the cytosol donors, there
were no significant sex-dependent difference
in the mean activity values, although the
degree of variation was greater in the females
(7.4-fold) than in the males (4.1-fold).
Hence, although the degree of variation in
GST activity (3- to 8-fold) is significantly less
than that observed with P450 activity (10- to
20-fold), this may be another important factor to account for in a human health risk
assessment for TCE.
Extrapolation of Experimental
Data for Human Health Risk
Assessment
Extrapolation from Animals to Humans
A major issue in the extrapolation of experimental metabolism data from animals to
humans involves the concentrations of
toxicants used. Animal studies often use relatively high concentrations of chemicals that
are severalfold to several orders of magnitude
higher than those to which humans may be
exposed either occupationally or environmentally. This, of course, brings into question the
relevance of such data. Examples of a number
of species-dependent differences in TCE
metabolism have been cited above.
Lipscomb et al. (66) recently addressed
this specific issue by characterizing the kinetics
and P450 isoform specificity of TCE metabolism to CH in liver microsomes from rats,
mice, and humans using a full range of concentrations of TCE, including those that are
estimated to occur in the livers of some occupationally exposed humans. Analysis of the
kinetics of TCE metabolism to CH showed
that rats metabolized TCE very differently
than either mice or humans: Rat liver microsomes exhibited at least three kinetically distinct processes for TCE metabolism, whereas
mouse and human liver exhibited only a single
process. Human liver microsomes exhibited
similar overall Km values for TCE as those
from rats or mice but had markedly (3- to 8fold) lower Vmax values. Hence, different
kinetic behavior among experimental animal
species and humans must be considered when
extrapolating animal data.
Extrapolation from in Vitro to in Vivo
The other most common type of extrapolation performed is from in vitro data to the
situation. This is particularly important for humans as most of our data come
in
vivo
from biopsy tissue samples, cell lines, or tissue
obtained from hospitals where the tissue cannot be used for transplant. One simple issue
is how to use data from liver microsomes or
isolated hepatocytes to quantitate metabolism
at the level of the entire organ or organism.
This process uses what have been called scaling factors, and data from two studies are
summarized (Table 18).
Lipscomb et al. (11) calculated parameters
that enable one to extrapolate to whole liver
in humans from measurements made in isolated hepatocytes or suspensions of microsomes. The parameters express the number of
hepatocytes per gram of liver, the amount of
microsomal protein per gram of liver, and the
amount of microsomal protein in a suspension of hepatocytes. Carlile et al. (160) made
similar use of scaling factors in studies with
rats to relate rates of drug metabolism in
various in vitro systems to rates in whole liver.
Kedderis (161) raised an important caveat
in the extrapolation of in vitro data to the in
vivo situation. Using enzyme induction as a
model, Kedderis showed that pretreatment
with inducers may significantly increase rates
of drug metabolism in vitro but that physiological constraints may dampen these effects
in vivo. He cites the example of furan
metabolism, which is catalyzed by CYP2E1.
Severalfold variations were observed in the
bioactivation of the hepatotoxicant furan by
isolated human hepatocytes owing to variations in this P450 isoform in the human population. Extrapolation of these data to the in
vivo situation showed that furan metabolism
is limited by hepatic blood flow. Because of
this limitation, variations in CYP2E1 content
among individual liver donors and increases
in Vmax due to inducers will be markedly
dampened in vivo.
Relative Roles of Oxidative
and GSH Conjugation
Pathways in TCE Metabolism
A principal issue in understanding the relative roles of the two major pathways of TCE
metabolism is that of pathway affinity. Based
on known kinetic parameters of TCE metabolism, relative rates of oxidative metabolism
and GSH conjugation under different exposure conditions (i.e., acute or chronic high
Table 18. Parameters used to extrapolate from microsomes and isolated hepatocytes to whole liver in humans.
Value
Parameter
Human livera
1 16 x 106 cells/g liver
Hepatocyte density
20.88 mg microsomal protein/g liver
Microsomal protein density: whole liver
179 pg microsomal protein/i 06 cells
Microsomal protein density: isolated cells
Rat liverb
2.6-2.7 mg DNA/g liver
DNA content of liver
21.5-26.8 pg DNA/1 06 cells
DNA content of hepatocytes
73-94 x 106 cells/g liver
Hepatocyte density (based on DNA data)
163-176 mg/g liver
Proteinc content of liver
1.4-1.5 mg protein/1 06 cells
Proteinc content of cells
109-125 x 106 cells/g liver
Hepatocyte density (based on protein data)
aData are from (11). bData are from Carlile et al. (160) and involve animals that have been treated with inducers of various P450s.
CTotal cellular protein, not just microsomal protein.
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
195
LASH ET AL.
doses versus chronic low doses, such as those
found as environmental contaminants) can
be determined. From this information, the
predominance of different metabolites at various doses can be discerned. Moreover,
knowledge of which metabolic pathway predominates at specific doses can help determine target organ specificity and mode of
action at those doses. Additionally, the two
major pathways of TCE metabolism are
known to have markedly different kinetics,
so that they saturate at different doses.
Hence, the relative proportion of metabolites
formed will likely vary markedly at low versus high doses. Specifically, CYP2E1 saturates at significantly lower doses of TCE than
do the GSTs, suggesting that flux of TCE
through the GSH conjugation pathway may
only be quantitatively important at higher
doses of TCE. As mentioned earlier, however, the GSH conjugation pathway, and
specifically the i-lyase reaction, generates
chemically reactive metabolites; formation of
even a small amount of a reactive species may
be sufficient to produce alterations in cellular
function whereas formation of relatively large
amounts of a non-reactive metabolite may
not produce any cellular dysfunction.
Hence, although one may conclude by
intuition that a low-affinity pathway may
only be of toxicological importance at high
doses and/or when the high-affinity pathway
is saturated, this is not necessarily correct for
a pathway such as that catalyzed by the ,1lyase that generates chemically unstable and
reactive metabolites. It has been argued that
the ,B-lyase pathway is not quantitatively
important for humans, based on the following points: a) Kinetic parameters (i.e., affinity
and capacity) of the I-lyase in human kidney,
as compared to those for P450 oxidation in
human liver, are such that P-lyase activity will
only become quantitatively significant at
extremely high exposure levels; b) TCE exposure studies comparing excretion of mercapturate to that of TCA and TCOH show that
the P450-derived metabolites are recovered in
urine at levels that are two to three orders of
magnitude higher than the GSH-derived
metabolite; and c) questions have arisen about
the relevance of reports of kidney tumors in
human populations exposed to TCE. I will
discuss the first two points below. The third
point will be only briefly discussed, as this is
dealt with in greater detail in the kidney
mode of action review (89).
Concerning the first point about the
relative kinetic parameters of the oxidative
and GSH conjugation pathways, there is no
doubt that flux through the P450 pathway is
much faster and that the total capacity is
much greater than for the GSH conjugation
and P-lyase reactions. There are two major
differences between the nature of the
96
metabolites formed by the two pathways.
First, the various enzymes of the GSH conjugation pathway and the membrane transporters for GSH and cysteine conjugates and
mercapturates exhibit significant tissue heterogeneity so that metabolites are directed to
the kidneys for intracellular accumulation
and metabolism to reactive species. Second,
the metabolites from the GSH conjugation
pathway that are believed to be responsible
for effects in the kidneys are chemically
unstable and highly reactive whereas those
derived from the oxidative pathway are relatively stable. Because of the nature of reactive
metabolites, it is likely that relatively small
amounts of these compounds can produce
significant biochemical and functional
changes in renal cells. One cannot, therefore,
directly extrapolate from amount of metabolite formed or pathway flux to toxicity.
Concerning the second point about
recovery of mercapturates compared to the
recovery of TCA and TCOH in the urine,
two types of exposures were conducted by
Henschler's group (96,97) that show the
variability in recovery of excreted metabolites
with exposure route (Table 19). However,
even in the studies that show lower ratios of
oxidative metabolites (TCA or TCA +
TCOH) to NAcDCVC, it has been argued
that ratios as little as 10:1 indicate that the
flux through the GSH conjugation pathway
is at least an order of magnitude slower than
that through P450. It must be emphasized,
however, that measurement of urinary mercapturate provides only an indication of a
portion of the GSH-dependent pathway and
that this portion is not related to that which
is associated with nephrotoxicity and nephrocarcinogenicity. Measurement of urinary
mercapturates, therefore, can give an indication of exposure and that some flux through
the GSH conjugation pathway has occurred.
It cannot, however, give an indication of total
flux through the GSH conjugation pathway
or through that portion of the pathway that
leads to effects on renal function. Moreover,
formation of even a small amount of reactive
species may be sufficient to produce toxic
effects or changes in intracellular regulatory
mechanisms.
Concerning the third point about the
occurrence of kidney tumors being much
lower than those in the other target organs
and this relating to a small flux through the
GSH conjugation and P-lyase pathways, the
following points should be noted: a) the
background rate of kidney tumors is
extremely small in humans and most animal
species studied (although some strains of rats
show a relatively high background rate); b)
the kidneys are particularly susceptible to
cytotoxic injury from even brief, low-dose
exposures to nephrotoxicants; and c) proximal
tubular epithelial cells can undergo a program
of apoptosis and regeneration after exposures
to sublethal doses, or even necrotic doses, of
nephrotoxicants. These points should highlight the difficulties in assessment of factors
involved in induction of renal carcinogenesis.
Although species- and sex-dependent differences are found in various steps of TCE
metabolism, it is important to also consider
the entire pathway as a whole and not just
individual reactions. Although the rate of a
specific step may be relevant to a consideration of toxicity if the toxicity is solely
dependent on the formation of a specific
metabolite, the balance between metabolite
formation and subsequent metabolic steps
and species differences in the ratio of these
steps are often of more importance.
In summary, it is clear that the oxidative
pathway is quantitatively the more important of the two major pathways of TCE
metabolism. However, the interorgan patterns of the GSH-dependent pathway and
the chemical nature of the critical metabolites generated by the two pathways greatly
alters the importance of simple comparisons
of quantitation. Furthermore, we do not
really have an accurate method to assess flux
through all branches of the GSH-dependent
pathway because chemically reactive species
are generated.
Table 19. Summary of studies on mercapturate and
oxidative metabolite excretion in urine of rats, mice, and
humans exposed to TCE.
Study l: Exposure of Wistar rats and NMRI mice to TCE
22 hr after exposure (50 mg/kg by oral gavage) (97)
Sex/species
Male rat
Female rat
Male mouse
Female mouse
TCA/NAcDCVC in urine
22.0
39.4
15.3
72.5
Study Il: Exposure of workers to TCE by inhalation or
dermal contact (97).
Worker number
1
5
6
8
TCA/NAcDCVC in urine
18.5
17.9
3.1
32.8
Study Ill: Exposure of humans and male and female
Wistar rats to TCE (6-hr inhalation). Cumulative
excretion of metabolites measured over 48 hr postexposure (96).
[TCE] in exposure
(TCA + TCOH)/
Species
chamber
NAcDCVC
Human
40 ppm
3,292
80 ppm
4,797
160 ppm
7,163
Rat
40 ppm
986
80 ppm
1,300
160 ppm
2,562
Environmental Health Perspectives
*
Vol 108, Supplement 2
*
May 2000
METABOLISM OF TCE
Questions and Research Needs
This section briefly discusses some key
research needs in the area of TCE metabolism. Although study of these questions is
not, in most cases, essential to completion of
an updated human health risk assessment for
TCE, research on these questions will answer
important questions and address uncertainties that will improve our understanding of
TCE metabolism.
Role of an Epoxide Intermediate
in TCE Metabolism
The question of whether TCE, like many
other halogenated solvents that undergo
P450-dependent metabolism, does so
through intermediate formation of an epoxide is still uncertain and should be resolved by
careful chemical studies.
Enzymology of DCA Formation
Two issues regarding amounts of and
sources of DCA formation are still incompletely resolved. The first issue relates to the
recovery of DCA in biological incubations
with TCE, as questions remain about the
recovery of DCA in tissue incubations and
biological fluids such as plasma and urine.
The second issue relates to the sources of
and specific enzymes involved in DCA formation. It is unclear what factors determine
how much of DCA is derived from TCA,
TCOH, or CH and what the specific
involvement is of enzymes such as P450s or
aldehyde dehydrogenase.
P450 Isoforn Activity Distribution
Although CYP2E1 has been identified as the
most active P450 isoform responsible for
oxidative metabolism of TCE, questions
remain about the potential activities of other
isoforms, both in uninduced and in induced
individuals. Expression of many P450 isoforms exhibits sex and species differences.
These differences, specifically in relation to
TCE metabolism, need to be more completely assessed as various physiological or
pathological changes in P450 isoform activity
distribution can alter metabolism and susceptibility to toxicity. Unequivocal identification
of the role of specific isoforms will require the
use of inhibitory antibodies and studies with
expressed and purified enzymes.
Role of Renal P450 in TCE Metabolism
and Bioactivation
Little investigation of renal P450 activities in
comparison to hepatic P450s has been undertaken. Although many of the same isoforms
as are found in the liver have been identified
in the kidneys, their regulation may be different in the two organs. For example, many of
the known inducers of hepatic CYP2E1 do
not have the same qualitative or quantitative
effects on renal CYP2E1. Based on the
knowledge that CYP2E1 is active in TCE
oxidation, this differential regulation should
impact on oxidative metabolism of TCE in
the kidneys.
Almost all of the focus of TCE metabolism in the kidneys has been on the GSHdependent pathway. Although there is no
direct evidence to suggest that intrarenal formation of metabolites such as TCA or DCA
is involved in the renal effects of TCE, this
issue has not been investigated fully so we
should not a priori rule it out.
GST Isozyme Distribution
andVariation
Recent work has revealed that the various
GSTs exist in isozymic forms and that sex-,
species-, and tissue-dependent differences
often exist. The substrate specificity of these
isozymes and specifically the activity of each
isozyme toward TCE is unknown at present.
Briining et al. (162) have recently shown that
genetic polymorphisms exist in two GSTs,
GSTM1 and GSTT1, and that differences in
expression of these isozymes may have toxicological significance for TCE bioactivation by
this pathway. Hence, work is needed to
define the isozyme composition in various
species, including humans, and in various tissues in each species, and to define the activity
of each isozyme towards TCE.
Quantitative Importance of DCVC
Metabolism in Humans
The difficulties in assessing the quantitative
importance of flux through the GSH conjugation pathway, and through the ,-lyase reaction specifically, have been discussed above in
some detail. Three key issues are
* Improved measurements of overall flux of
TCE through the GSH conjugation
pathway and the f-lyase as compared to
that through the oxidative pathway by
more complete determinations of rates of
metabolism and kinetic parameters in tissues from various species, particularly
humans.
* Development of more accurate, noninvasive means of measuring flux through the
3-lyase pathway in humans. Urinary mercapturates only provide information on
stable end products and these do not necessarily correlate with formation of reactive
metabolites that produce nephrotoxicity
and/or nephrocarcinogenicity.
* Data on formation of covalent adducts of
DCVC with DNA and protein have been
difficult to interpret and may not directly
correspond to cytotoxic or tumorigenic
events. Hence, additional studies on the
functional significance of covalent binding to cellular macromolecules are
warranted.
Environmental Health Perspectives * Vol 108, Supplement 2 * May 2000
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