Assessing the health risks following environmental exposure to hexachlorobutadiene

Article abstract of DOI:10.1016/S0378-4274(02)00372-7

Hexachloro-1,3-butadiene (HCBD) has been reported to be toxic to the rat kidney in a 2 year study at doses higher than 0.2 mg/kg/day. The toxicity is known to be a consequence of the metabolism of HCBD by glutathione conjugation and the renal β-lyase pathway. Neither toxicity data, nor data on the metabolism of HCBD, are available in humans. In the current work, the potential of HCBD to cause kidney damage in humans environmentally exposed to this chemical has been assessed quantitatively by comparing the key metabolic steps in rats and humans. To that end, the hepatic conjugation of HCBD with glutathione, the metabolism of the cysteine conjugate by renal β-lyases and N-acetyltransferases, and the metabolism of the N-acetylcysteine conjugate by renal acylases has been compared in vitro in rat and human tissues. Rates for each metabolic step were lower in humans than in rats; 5-fold for glutathione conjugation, 3-fold for β-lyase and 3.5-fold for N-acetyltransferase. Acylase activity could not be detected in human kidney cytosol. Use of these data in a physiologically based toxicokinetic model to quantify metabolism by the β-lyase pathway demonstrated that metabolism in humans was an order of magnitude lower than that in rats. At the no effect level for kidney toxicity in the rat the concentration of β-lyase metabolites was calculated by the model to be 137.7 mg/l. In humans the same concentration would be achieved following exposure to 1.41 ppm HCBD. This is in contrast to the figure of 0.6 ppb which is obtained when it is assumed that the risk is associated with the internal dose of HCBD itself rather than β-lyase metabolites.

Full text of DOI:10.1016/S0378-4274(02)00372-7

Toxicology Letters 138 (2003) 63ꢁ73
/
www.elsevier.com/locate/toxlet
Assessing the health risks following environmental exposure to
hexachlorobutadiene
Trevor Green a,*, Robert Lee a, David Farrar b, James Hill c
a Syngenta Central Toxicology Laboratory, Alderley Park, Macclesfield, Cheshire SK10 4TJ, UK
b Ineos Chlor, The Heath, Runcorn, Cheshire, UK
c ICI Regional and Industrial Businesses, The Heath, Runcorn, Cheshire, UK
Received 12 January 2002; accepted 17 June 2002
Abstract
Hexachloro-1,3-butadiene (HCBD) has been reported to be toxic to the rat kidney in a 2 year study at doses higher
than 0.2 mg/kg/day. The toxicity is known to be a consequence of the metabolism of HCBD by glutathione conjugation
and the renal b-lyase pathway. Neither toxicity data, nor data on the metabolism of HCBD, are available in humans. In
the current work, the potential of HCBD to cause kidney damage in humans environmentally exposed to this chemical
has been assessed quantitatively by comparing the key metabolic steps in rats and humans. To that end, the hepatic
conjugation of HCBD with glutathione, the metabolism of the cysteine conjugate by renal b-lyases and N-
acetyltransferases, and the metabolism of the N-acetylcysteine conjugate by renal acylases has been compared in
vitro in rat and human tissues. Rates for each metabolic step were lower in humans than in rats; 5-fold for glutathione
conjugation, 3-fold for b-lyase and 3.5-fold for N-acetyltransferase. Acylase activity could not be detected in human
kidney cytosol. Use of these data in a physiologically based toxicokinetic model to quantify metabolism by the b-lyase
pathway demonstrated that metabolism in humans was an order of magnitude lower than that in rats. At the no effect
level for kidney toxicity in the rat the concentration of b-lyase metabolites was calculated by the model to be 137.7 mg/l.
In humans the same concentration would be achieved following exposure to 1.41 ppm HCBD. This is in contrast to the
figure of 0.6 ppb which is obtained when it is assumed that the risk is associated with the internal dose of HCBD itself
rather than b-lyase metabolites.
# 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Hexachlorobutadiene; Kidney; Modelling; Risk assessment
1. Introduction
ered at an historic waste disposal site in the UK.
The transfer of vapour from the site into the
surrounding strata led to the detection of low
concentrations of HCBD, typically less than 8
ppb, in the indoor air of a small number of houses
immediately adjacent to the site. The consequences
of this exposure on human health are uncertain,
there being very little information on the toxico-
Environmental contamination with hexachloro-
1,3-butadiene (HCBD) has recently been discov-
* Corresponding author. Tel.: ꢀ
1625-586396
E-mail address: trevor.green@syngenta.com (T. Green).
/
44-1625-515458; fax: ꢀ44-
/
0378-4274/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 3 7 8 - 4 2 7 4 ( 0 2 ) 0 0 3 7 2 - 7

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T. Green et al. / Toxicology Letters 138 (2003) 63ꢁ73
/
logical effects of HCBD derived from studies on
humans. Consequently, an assessment of the
possible risks to human health has to be based
on laboratory animal data.
HCBD has the potential to be nephrotoxic in
humans. That potential can be assessed quantita-
tively by comparing the key metabolic steps in the
activation of HCBD with those in the rat where
the toxicity is known. To that end the hepatic
conjugation of HCBD with glutathione, the meta-
bolism of the cysteine conjugate by renal b-lyases
and N-acetyltransferases, and the metabolism of
the N-acetylcysteine conjugate by renal acylases
have been compared in vitro in rat and human
tissues. The data have then been used in a
physiologically based toxicokinetic model (PB-
TK model) to quantify the extent of metabolism
by b-lyase in order to assess the risks to humans
exposed to HCBD. This approach has been
compared with that using default assumptions in
the absence of metabolic data. A summary of the
metabolism of HCBD and the key steps measured
in vitro and used in the model is shown in Fig. 1.
The toxicity of HCBD has been studied exten-
sively in the rat and to a lesser extent in a number
of other laboratory species. The primary target
organ in all species is the kidney. In the rat, the
nephrotoxicity is well characterised with respect to
dose, morphological changes and changes in blood
and urine biochemistry. Following either a single
oral or intraperitoneal dose, necrosis is seen in the
pars recta of the proximal tubule (Ishmael et al.,
1982) together with increases in blood urea nitro-
gen and marked increases in urinary protein,
glucose and a number of enzymes normally found
in the proximal tubule of the kidney (Lock and
Ishmael, 1979; Nash et al., 1984; Ishmael and
Lock, 1986). Following chronic dosing, a low
incidence of kidney tumours was found in rats
administered HCBD in the diet. The no observed
effect level (NOEL) for kidney toxicity in this
study was 0.2 mg/kg/day.
2. Materials and methods
The mode of action of HCBD as a nephrotoxin
is also understood. HCBD is metabolised in the
liver to form a glutathione conjugate which is
subsequently further metabolised to the equivalent
cysteine conjugate (Nash et al., 1984; Wolf et al.,
1984). The cysteine conjugate may be metabolised
to its mercapturate, the N-acetylcysteine conjugate
2.1. Chemicals
HCBD (99%) was supplied by BDH Chemicals
Ltd, Poole Dorset. S-(1,2,3,4,4-pentachloro-1,3-
butadienyl)glutathione
(1,2,3,4,4-pentachloro-1,3-butadienyl)-
(HCBD-CYS) and N-acetyl-S-(1,2,3,4,4-penta-
chloro-1,3-butadienyl)- -cysteine (HCBD-NAC)
were synthesised as described previously (Nash et
al., 1984). [Acetyl-1-14C]-coenzyme A (40ꢁ
60 mCi/
(HCBD-GSH),
S-
L-cysteine
or undergo CÃ
/
S bond cleavage by renal CÃ
/
S or b-
L
lyase enzymes (Jaffe et al., 1983; Green and Odum,
1985; Schrenk et al., 1988). It is the action of b-
lyases that leads to the formation of a number of
reactive intermediates and the observed kidney
toxicity. The N-acetylcysteine conjugate may be
excreted in urine or converted back to the cysteine
conjugate by the action of acylases, the latter
increasing the amount of cysteine conjugate avail-
able for cleavage by b-lyases. Nephrotoxicity is
therefore directly linked to b-lyase activity and the
amount of substrate, the cysteine conjugate, avail-
able to this enzyme.
/
mmol) was obtained from Amersham-Pharmacia,
Cardiff, UK.
2.2. Animals and tissues
Male Alderley Park (Alpk/APfSD) rats weigh-
ing 180ꢁ200 g were obtained from the Barriered
/
Animal Breeding Unit, Alderley Park, Maccles-
field, Cheshire, UK. The clinical condition of all
animals was monitored prior to the start of the
experiments and any animal showing adverse
clinical signs was removed.
The animals received Rat and Mouse Number 1
pelleted diet from Special Diet Services, Witham,
Essex, UK, and mains water ad libitum. The
The toxicity of HCBD in humans is not known,
nor has the metabolism of HCBD been studied
either in vivo or in vitro. All of the relevant
enzymes needed to activate HCBD, including b-
lyases, are known to exist in humans and hence

T. Green et al. / Toxicology Letters 138 (2003) 63ꢁ
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65
Fig. 1. The metabolic activation of HCBD. The enzymes shown are those compared between rats and humans and used in the PB-TK
model.
environment of the animal room was controlled
with the temperature being maintained within a
tained from the UK Human Tissue Bank,
Leicester, UK. The donors were 4 males (age
54ꢁ73) and 2 females (ages, 41 and 51 years).
/
target range of 19ꢁ
/
23 8C, relative humidity within
Causes of death were cardiac arrest (3), cerebral
oedema/haemorrhage (2) and cerebral tumour.
a target range of 40ꢁ
/
70%, an artificial light cycle
of 12 h light and 12 h darkness, and between 25
and 30 air changes per hour. All conditions were
monitored by the Honeywell site monitoring
system and any excursions outside these ranges
were noted by means of an external alarm and
recorded.
2.3. In vitro metabolism
2.3.1. Preparation of tissue fractions
Animals were sacrificed by asphyxiation with a
rising concentration of CO2 followed by cardiac
puncture. The livers and kidneys were removed,
pooled and thoroughly washed in ice cold 1.15%
potassium chloride. The tissues were scissors
minced, and homogenates, 30% (w/v) for liver
and 10% (w/v) for kidney, prepared in 0.25 M
sucrose, 20 mM tris, 5 mM EDTA pH 7.4 using a
Potter Elvehjem homogenizer. The homogenates
Human liver samples (nꢂ3) were obtained from
/
the Queen Elizabeth Hospital, Birmingham, UK
and were from fresh liver excess to transplantation
requirements. The donors were male caucasians
age 41ꢁ57 years, the causes of death were cardiac
/
arrest (2) and intracranial bleed. Samples were
provided in compliance with local ethical guide-
lines. Human kidney samples (nꢂ6) were ob-
/

66
T. Green et al. / Toxicology Letters 138 (2003) 63ꢁ73
/
were centrifuged at 16 900ꢃ
the supernatants transferred to fresh tubes and
centrifuged at 105 000ꢃg for 70 min at 4 8C. The
supernatant (cytosol) was removed from the
microsomal pellet and stored at ꢄ70 8C. The
/
g for 15 min at 4 8C,
the HCBD (in 5 ml DMSO). The reaction was
terminated at 60 min by the addition of 1 ml 10%
(w/v) trichloroacetic acid. The tubes were centri-
/
fuged at 3 000ꢃg to remove protein, and the
/
/
sample neutralised by the addition of 0.5 ml 1 M
sodium hydroxide.
The samples were analysed for HCBD-GSH
microsomal pellet was resuspended in 20 mM tris,
1.15% potassium chloride pH 7.4, centrifuged at
105 000ꢃ
/
g for 70 min at 4 8C, and the pellet
resuspended in 0.1 M sodium phosphate pH 7.4
using a 5 mm Hypersil ODS 4.6ꢃ
/
200 mm
analytical column with a 4.6ꢃ30 mm guard
/
(2:1 w/v original tissue wet weight to buffer). The
column (Hichrom, UK) on a Shimadzu LC-10
system, with UV monitoring at 220 nm. The
glutathione conjugate was eluted in 10% acetoni-
trile (0.025% trifluoroacetic acid)/90% water
(0.025% trifluoroacetic acid) at 1 ml/min using a
gradient, which after rising to 75% acetonitrile
over 20 min, rose to 100% acetonitrile over a
further 2.5 min. Quantification was performed
using HCBD-GSH standards. The HCBD-GSH
standard eluted at 17.8 min.
microsomes were stored at ꢄ70 8C until required.
/
For the preparation of kidney mitochondrial
fractions, the tissues were scissors minced, and a
6.7% (w/v) homogenate was prepared in 0.25 M
sucrose, 10 mM tris pH 7.4 using a Potter
Elvehjem homogenizer. The homogenates were
centrifuged at 600ꢃ
pellet discarded, and the supernatent centrifuged
for a further 10 min at 600ꢃg. The supernatant
was again removed and centrifuged at 10 000ꢃ
/
g for 10 min at 4 8C, the
/
/g
for 20 min at 4 8C. The supernatant was now
discarded, and the pellet washed in 0.25 M
sucrose, 10 mM tris pH 7.4, before centrifuging
2.3.3. Renal b-lyase
Cysteine conjugate b-lyase was measured in rat
and human kidney cytosol and mitochondria using
the method of Stevens and Jakoby (1983).
For the determination of cytosolic b-lyase
/
at 10 000ꢃg for 20 min at 4 8C. The mitochon-
/
drial pellet was resuspended in 0.25 M sucrose, 10
mM tris pH 7.4 (1:6.67 w/v original tissue wet
activity, incubation mixtures contained 0.025ꢁ
weight to buffer) and stored at ꢄ
required.
/
70 8C until
1.25 mM HCBD-CYS, 0.1 mM NADH, 0.1 units
LDH, and kidney cytosol (1.3 mg/ml) in a final
volume of 1 ml of 50 mM potassium phosphate,
pH 7.4 at 37 8C. The mixture was pre-incubated
for 4 min prior to the initiation of the enzyme
reaction by the addition of HCBD-CYS. Pyruvate
production over a 30 min period (60 min for the
human samples), monitored spectrophotometri-
cally at 340 nm, was used as a measure of enzyme
activity.
The human liver and kidney samples were
processed as above, except that they were homo-
genised using an Ultra Turrux homogenizer, and
the microsomal pellets were resuspended in 0.25 M
sucrose, 20 mM tris, 5 mM EDTA pH 7.4 (1:1 w/v
original tissue wet weight to buffer).
Protein concentrations in the tissue fractions
was determined using the method of Lowry et al.,
1951
For the determination of mitochondrial b-lyase
activity, incubation mixtures contained 0.025ꢁ1.75
/
2.3.2. Hepatic glutathione conjugation
mM PCBD-CYS, mitochondria (1.0 mg/ml for rat
and 1.75 mg/ml for humans) in a final volume of 1
ml of 50 mM potassium phosphate, pH 7.4 at
37 8C. The mixture was pre-incubated for 4 min
prior to the initiation of the enzyme reaction by
the addition of the mitochondria. The incubation
was terminated after 5 min by the addition of 0.2
ml 10% trichloroacetic acid. The samples were
The glutathione S-transferase catalysed meta-
bolism of HCBD was determined by measuring
the formation of HCBD-GSH.
The incubation mixtures contained 0.01ꢁ0.4
/
mM HCBD, 5 mM glutathione, and hepatic
microsomes (1 mg), in a final volume of 1 ml 100
mM potassium phosphate buffer, pH 7.4 at 37 8C.
The tubes were preincubated at 37 8C for 4 min
before the reaction was initiated by the addition of
spun at 1000ꢃ
/
g, for 10 min at 4 8C. 0.654 ml of
the supernatant was neutralised by 0.196 ml 1 M

T. Green et al. / Toxicology Letters 138 (2003) 63ꢁ
/73
67
potassium hydrogen phosphate (K2HPO4). En-
zyme activity was measured by determining the
conversion of the pyruvate formed to lactate using
0.2 mM NADH, and 0.1 units LDH.
diluted to 2.5 ml with 50 mM potassium phos-
phate, pH 7.4, and the amount of product formed
was measured fluorometrically using excitation at
390 nm, and emission at 475 nm with a band width
of 4 nm. The amount of product formed was
quantified against a HCBD-CYS standard.
2.3.4. Renal N-acetyl transferase
Cysteine N-acetyl transferase activity was de-
termined by measuring the amount of labelled
acetyl coenzyme A conjugated to HCBD-CYS
using the method of Duffel and Jakoby (1982).
2.4. PB-TK modelling
A PB-TK model was constructed using Ad-
vanced Continuous Simulation Language (ACSL
Incubation mixtures contained 0.05ꢁ
/
1.0 mM
HCBD-CYS, 0.8 mM [acetyl-1-14C]-coenzyme A
*
/
Mitchell Gauthier Associates Inc, Concord, MA)
software and was based on the inhalation model
described by Ramsey and Andersen (1984), with
the addition of a kidney compartment. Physiolo-
gical parameters used in the model were taken
from standard texts and metabolic rates were those
determined experimentally as described above.
Partition coefficients for blood:air and blood:fat
were measured experimentally as described by
Gargas et al., 1989. The model was used firstly
to determine an inhalation dose in rats which gave
the same body burden as that following dietary
administration at the NOEL for kidney toxicity of
0.2 mg/kg/day. The concentration of b-lyase de-
rived metabolites in the kidney was then calculated
at this dose using the metabolic parameters.
Subsequently, the model was used to predict a
human exposure which gave the same concentra-
tion of b-lyase metabolites, in this case using the
human metabolic parameters and physiological
constants. Blood and fat concentrations of HCBD
were also predicted to determine whether HCBD
accumulated in humans upon continuous exposure
to environmental concentrations.
(0.25 mCi/mmol), and kidney microsomes (0.11 mg
for rat, and 0.09ꢁ0.15 mg for human), in a final
/
volume of 0.25 ml 0.2 M potassium phosphate, pH
7.0 at 37 8C. The mixture was preincubated for 2
min at 37 8C, prior to the initiation of the reaction
by the addition of the microsomes. The reaction
was terminated at 5 min by the addition of 0.75 ml
1.33 M acetic acid and 2 ml cyclohexanone. The
samples were vortexed and the layers separated by
centrifugation at 500ꢃg for 3 min. 0.5 ml aliquots
/
of the cyclohexanone layer were added to 10 ml
Ultima-Gold (Canberra Packard), and the radio-
activity determined by liquid scintillation count-
ing.
2.3.5. Renal acylase
The de-acetylation of HCBD-NAC to HCBD-
CYS was determined by measuring the amount of
de-acetylated product formed using the method of
Uttamsingh and Anders (1999).
Incubation mixtures contained 0.1ꢁ
HCBD-NAC, 0.1 mM aminooxyacetic acid, and
dialysed kidney cytosol (0.6ꢁ0.7 mg for rat, 1 mg
/
2.0 mM
/
for human) in a final volume of 1 ml of 50 mM
potassium phosphate, pH 7.4 at 37 8C. The
mixture was pre-incubated for 4 min prior to the
initiation of the reaction by the addition of the
HCBD-NAC. The reaction was terminated at 30
min (45 min for the human samples) by the
addition of 200 ml ice cold 20% trichloroacetic
2.4.1. Assumptions made in the model
Complete adsorption was assumed following
dietary administration of HCBD and this route
was not therefore described in the model. The
model was used to calculate a continuous exposure
concentration which gave a daily body burden of
0.2 mg/kg HCBD. The partition coefficients which
were considered to have the greatest impact on the
output of the model, air:blood and blood:fat, were
measured experimentally. Blood:tissue compart-
ment partition coefficients for small lipophilic
molecules normally approximate to unity (Gargas
acid. The samples were then spun at 1000ꢃg for
/
10 min at 4 8C. 75 ml of the supernatant was
neutralised with 30 ml 1 M K2HPO4 and mixed
with 100 ml 0.2 M borate buffer, pH 8.5. Fluor-
escamine (0.1 mg in 100 ml of acetone) was then
added with mixing. After 10 min the samples were

68
T. Green et al. / Toxicology Letters 138 (2003) 63ꢁ73
/
et al., 1989) and it was assumed that this was the
case for HCBD. As such these values have little
impact on the output of the model.
than that found in human liver. The affinity
constant (Km) for the reaction was approximately
1.3-fold higher in the rat than in the human
samples. Less than a 2-fold variation was found
in either the human Km or Vmax for this reaction
(Table 1).
Glutathione conjugation of HCBD is catalysed
by both microsomal and cytosolic glutathione S-
transferases. The microsomal rate is 40-fold higher
in human liver, and 3.4-fold higher in rat liver,
than those found in the equivalent cytosolic
fractions (Oesch and Wolf, 1989) and in the model,
hepatic metabolism of HCBD is based on the rates
measured in microsomal fractions. Metabolism of
HCBD by glutathione conjugation is also known
to occur in the kidney. However, the rates in the
kidney are 15-fold lower than those in the liver on
a mg protein basis and 90-fold lower on a whole
organ basis (Morganstern et al., 1984). Conse-
quently, in the model, glutathione conjugation
only occurs in the liver.
In the rat, the glutathione conjugate of HCBD is
known to be excreted in bile and to undergo
enterohepatic recirculation (Nash et al., 1984;
Reichert et al., 1985). This information is not
available in humans, either quantitatively or
qualitatively. In the absence of this information
it is assumed, in both species, that all of the
glutathione conjugate formed in the liver is avail-
able, as the cysteine conjugate, for further meta-
bolism by the kidney. In the kidney, metabolism
occurs by b-lyase, N-acetyltransferase and acylase.
b-lyase metabolism is based on the rates in
mitochondria since these were higher than cyto-
solic rates in the rat and a rate was not detectable
in human cytosol. Furthermore, the mitochon-
drion is believed to be the target organelle for b-
lyase activated nephrotoxins (Bruschi et al., 1998).
The mercapturic acid (HCBD-NAC) is also elimi-
nated in urine. Urinary excretion is based on that
reported for the rat (Nash et al., 1984; Reichert et
al., 1985).
The metabolism of HCBD-CYS by renal b-
lyases is shown in Table 2. A metabolic rate could
not be detected in the human kidney cytosol
samples. Based on the limit of detection of the
assay, the rate is human kidney cytosol is at least
23-fold lower than that in the rat. Mitochondrial
fractions were significantly more active than
cytosolic fractions, the rate in rat kidney mito-
chondria being approximately 4-fold greater than
that in the cytosol fraction. A significant rate,
which was 3-fold lower than that in the rat, was
also detected in human kidney mitochondria. The
affinity constants Km for the mitochondrial en-
zyme were essentially similar between the two
species. In humans, the variability between indivi-
duals was slight, between 2- and 3-fold.
Both rat and human kidney were highly active
for the metabolism of HCBD-CYS to its N-
acetylcysteine conjugate (Table 3). The rates
(Vmax) were 27-fold greater in the rat and 21-
fold greater in humans than the maximum rates
for the competing b-lyase reactions in cytosolic
and mitochondrial fractions combined.
A significant rate was detected for the metabo-
lism of HCBD-NAC by acylases in rat, but not
human kidney fractions (Table 3). Based on the
limit of detection of the assay, the rate in humans
was 74-fold lower than that in the rat.
Table 1
The conjugation of HCBD with glutathione in rat and human
liver microsomes
3. Results
Species
Km (mM)
Vmax (nmol/min/mg)
Rat
0.21
0.16
0.16
0.16
0.16
1.23
0.22
0.19
0.34
0.25
3.1. In vitro metabolism
Human 90
Human 94
Human 98
The conjugation of HCBD with glutathione in
rat liver microsomal fractions occurred at a rate
(Vmax) which was approximately 5-fold higher
Humanꢁmean
/

T. Green et al. / Toxicology Letters 138 (2003) 63ꢁ
/73
69
Table 2
The metabolism of HCBD-CYS by b-lyases in rat and human kidney fractions
Species
Cytosol
Km (mM)
0.30
Mitochondria
Km (mM)
Vmax (nmol/min/mg)
1.13
Vmax (nmol/min/mg)
Rat
0.12
0.12
0.16
0.67
0.28
0.15
ND
0.25
4.17
0.90
1.43
1.78
1.56
0.73
1.74b
1.76
Human 003
Human 004
Human 005
Human 006
Human 007
Human 008b
ꢁ/
ꢁ/
ꢁ/
ꢁ/
ꢁ/
ꢁ/
ꢁ/
B
/
0.05a
0.05a
0.05a
0.05a
0.05a
0.05a
0.05a
B
/
B
/
B
/
B
/
B
/
Humanꢁ
/
mean
B/
a
No measurable rate, limit of detection shown.
Measured at a single substrate concentration of 1.25 mM.
b
3.2. PB-TK model parameters
3.3. PB-TK model predictions
The blood:air partition coefficients for rat and
human blood were 115 and 75 respectively. A
fat:air partition coefficient of 2717 was obtained
for rat fat and used in the model for both rat and
human fat. This gave fat:blood partition coeffi-
cients of 24 for the rat and 36 for human. The
metabolic constants used in the model were those
For the purposes of modelling the metabolism
of HCBD in rats and humans, it has been assumed
that exposures occur at concentrations of less than
1 ppm. At these exposure levels the concentrations
of HCBD found in tissues increase linearly with
increasing dose (Table 5). Fig. 2 illustrates the
blood and fat concentrations of HCBD in humans
exposed to 0.01 ppm, continuously for 10 days.
Steady state has been reached in both blood and
fat compartments by the end of exposure and post-
exposure the concentrations of HCBD decline with
half-lives of 6 h in blood and 26 h in fat. Because
steady state has been reached, blood and fat levels
shown in Tables 1ꢁ3 expressed in model units (mg/
/
h/kg tissue). Where metabolic rates could not be
detected (human acylase Table 3), the rate used in
the model was the limit of detection of the assay.
The values of physiological parameters used in the
model are shown in Table 4.
Table 3
N-Acetyl transferase and acylase activities in rat and human kidney fractions
Species
N-Acetyl transferasea
Acylaseb
Km (mM)
1.43
Km (mM)
Vmax (nmol/min/mg)
Vmax (nmol/min/mg)
7.36
Rat
0.39
0.21
0.20
0.10
0.05
0.24
0.13
0.19
144.9
40.8
8.8
Human 003
Human 004
Human 005
Human 006
Human 007
Human 008
ꢁ/
ꢁ/
ꢁ/
ꢁ/
ꢁ/
ꢁ/
ꢁ/
B
/
0.10a
0.10a
0.10a
0.10a
0.10a
0.10a
0.10a
B
/
25.6
27.5
60.6
62.5
37.6
B
/
B
/
B
/
B
/
Humanꢁ
/
mean
B/
a
Measured in microsomal fractions with HCBD-CYS as the substrate.
Measured in cytosol fractions with HCBD-NAC as the substrate.
b

70
T. Green et al. / Toxicology Letters 138 (2003) 63ꢁ73
/
Table 4
Partition coefficients and physiological parameters used in the
HCBD PB-TK model
Parameter
Rat
Human
Partition coefficientsa
Blood/air
Fat/blood
115
24
75
36
Tissue compartment*/% body weight
Liver
Kidney
4
1.1
2.6
0.44
21
Fat
Richly perfused
Slowly perfused
7
5
4.5
62
75
Fig. 2. Predicted concentrations of HCBD in human blood and
fat during and post-exposure to 10 ppb HCBD 24 h/day for 10
days. CA is the concentration of HCBD in arterial blood and
CF the concentration in fat.
Blood flow*% cardiac output
/
Liver
Kidney
Fat
25
17
5
24
18
5
Richly perfused
Slowly perfused
34
19
34
19
continuous exposure to 0.07 ppm/day. Exposure
at this concentration gave a renal concentration of
b-lyase metabolites of 137.7 mg/l. The NOEL for
kidney toxicity in the rat equates, therefore, to a
target organ dose of b-lyase derived metabolites of
137.7 mg/l. To produce the same concentration of
b-lyase metabolites in the human kidney, the
model calculates that humans would have to be
continuously exposed to 1.41 ppm HCBD.
As indicated above, a rate was not detectable for
the metabolism of HCBD-NAC by human kidney
acylase. Consequently, the limit of detection of the
assay was used as the metabolic rate for this
pathway in the model. If the rate for human
acylase is set to zero, the difference in b-lyase
metabolism between rats and humans increases
from approximately 20- to almost 50-fold.
Alveolar ventilation rate (l/h)
Cardiac output (l/h)
Body weight (kg)
5.4
5.4
348
348
70
0.25
a
Liver/blood, kidney/blood, slow/blood, rapid/blood coeffi-
cients were set to unity.
would not be expected to increase with continued
exposure beyond 10 days.
In order to assess the likelihood that kidney
toxicity would occur in humans following expo-
sure to HCBD, the model was first used to predict
metabolism by the b-lyase pathway in rats at the
NOEL of 0.2 mg/kg/day for kidney toxicity. In the
rat, a body burden of 0.2 mg/kg HCBD/day was
calculated by the model to be equivalent to
Table 5
PB-TK model predictions
Species
Rat
Exposure ppm
Blood level mg/l
Fat concentration mg/l
1
0.62
0.0062
4.16
14.9
0.148
5.80
0.01
0.01
Half-life (t1/2-h)
Human
1
0.68
0.0068
6.0
24.6
0.245
26.0
0.01
0.01
Half-life (t1/2-h)
Blood and fat levels in rats and humans continuously exposed to HCBD.

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71
4. Discussion
HCBD failed because of difficulties in maintaining
a stable atmosphere of this high boiling chemical.
This meant that the model was constructed using a
limited set of existing data and the in vitro studies
described here. Nevertheless, the human exposures
to HCBD were a real issue, they were a matter of
immediate concern and required a rapid resolution
of the associated risks. Thus, incorporation of the
data describing the complex metabolic pathway
into the risk assessment process was considered to
be a worthwhile improvement over the default
which is based on inhaled dose alone.
Assessment of the risk following continuous
exposure to very low levels of a chemical in the
environment is always problematical and can
normally only be estimated from laboratory ani-
mal data with the application of appropriate safety
margins to allow for the uncertainties involved.
Frequently, the animal data will have been
obtained at considerably higher dose levels with
different exposure scenarios and possibly even by a
different route of administration. The data avail-
able to assess the risks to humans from environ-
mental exposure to HCBD is typical. The primary
data source is a 2 year rat chronic toxicity and
carcinogenicity study which was conducted at a
maximum tolerated dose with the test material
administered in the diet (Kociba et al., 1977).
Thus, assessment of human risks requires extra-
polation across doses which differ by orders of
magnitude and extrapolation between dietary and
inhalation exposure. Currently, the only tool
available to make this type of extrapolation is
PB-TK modelling.
Although nothing is known about the toxicity of
HCBD in humans it is known that the enzymes
involved in its metabolic activation in the rat are
present in humans. Consequently, it must be
assumed that HCBD has the potential to be toxic
in the human kidney, as it is in the rat. HCBD
itself is not nephrotoxic, the effects in the kidney
are in fact induced by a metabolite produced at the
end of a metabolic cascade involving a number of
enzymes in more than one organ. Thus, in addition
to comparing uptake between the two routes of
exposure, a comparison of the metabolic cascade
that leads to a nephrotoxic metabolite is also
required in rats and humans.
The renal toxicity of HCBD is known to be a
consequence of the metabolism of the cysteine
conjugate of HCBD by renal b-lyases. The amount
of this conjugate available for metabolism by these
enzymes is determined by a number of factors.
These are principally the uptake and distribution
of HCBD itself, and the activities of those enzymes
involved in the formation and further metabolism
of the cysteine conjugate. In the PB-TK model the
uptake and distribution of HCBD is determined
using measured partition coefficients and standard
values for physiological parameters such as venti-
lation rates and blood flows. Metabolism is
described by four key enzymes which control the
formation, activation and further metabolism of
the cysteine conjugate of HCBD (Fig. 1). The
glutathione S-transferases catalyse the conjuga-
tion of HCBD with glutathione and determine the
amount of cysteine conjugate available for further
metabolism in the kidney. The cysteine conjugate
is a substrate for both renal b-lyases and N-
acetyltransferases. The b-lyases which activate
the cysteine conjugate, are pyridoxal phosphate
dependent enzymes and are only active with the
cysteine conjugate. Consequently, metabolism of
the cysteine conjugate by N-acetyltransferases to
form the mercapturic acid is a true detoxification
reaction. The fourth enzyme system, the renal
acylases convert the N-acetylcysteine conjugate
back to the cysteine conjugate thus making more
substrate available for the b-lyases. Each of these
processes has been included in the model.
Although PB-TK modelling is an ideal means of
incorporating these types of data into risk assess-
ments, there were a number of constraints during
the construction of the model used here. There are
no human metabolic or kinetic data of any type
available to validate the model, nor under the
circumstances is there any opportunity to generate
such data. Similarly, there are no data in animals
following inhalation exposure. Attempts to ex-
The b-lyases comprise a number of enzymes
which are able to cleave the CÃ
/
S bond of
haloalkene cysteine conjugates to give a reactive
thiol, pyruvate and ammonia (Abraham et al.,
posed rats to low concentrations (:1 ppm) of
/

72
T. Green et al. / Toxicology Letters 138 (2003) 63ꢁ73
/
1995; Stevens and Jakoby, 1983; Stevens et al.,
1988). The lyases are found in both the soluble
(cytosolic) and mitochondrial fractions of kidney
homogenates, the relative activities of the enzymes
vary according to the structure of the cysteine
conjugate. The HCBD conjugate is metabolised
mainly by the mitochondrial enzymes which is
consistent with the known toxicity of cysteine
conjugates of this type towards the mitochondrial
respiratory chain (Bruschi et al., 1998).
Two major forms of the N-acetyltransferases
are known, one of which (NAT2) is known to be
polymorphic in the human population. Which of
the two isoforms is responsible for the acetylation
of cysteine conjugates of this type is not known.
Both forms are found in the microsomal fractions
and hence total activity has been measured in the
current work. The variability between individuals
As noted, the only available toxicity data on
which to make a human health assessment is the
lifetime study in rats in which HCBD was admi-
nistered in the diet for 2 years (Kociba et al., 1977).
In that study, the NOEL for kidney pathological
change was 0.2 mg/kg/day. In the absence of the
metabolic data described in this paper data, the
UK Committee on Toxicity (COT, 2000) used the
toxicity data and default assumptions to calculate
safety margins. The uptake of HCBD from inhaled
air was assumed to be complete, and humans were
considered to be as sensitive as the rat to the toxic
or carcinogenic effects of HCBD. This approach
calculates that a gavage dose of 0.2 mg/kg/day is
equivalent to an inhaled dose of 60 ppb. Their
application of a safety margin of 100 to account
for cross-species and intra-species variability for
non-carcinogenic and reproductive effects resulted
in an exposure of 0.6 ppb which they regarded as
being without appreciable adverse health effect
(COT, 2000). When the metabolic data described
above is used in a PB-TK model, the concentration
of b-lyase metabolites in the rat kidney at the
NOEL of 0.2 mg HCBD/kg/day is calculated to be
137.7 mg/l. To achieve the same concentration in
humans would require continuous exposure to
1.41 ppm HCBD which is equivalent to an uptake
of 2.0 mg/kg/day. Applying the same 100-fold
safety margin (COT, 2000) results in an exposure
of 14.1 ppb which can be regarded as being
without appreciable adverse health effect. This is
in contrast to the figure of 0.6 ppb obtained using
default assumption without the species specific
metabolic data.
was relatively low, 25.6ꢁ62.5 nmol/min/mg, with a
/
single sample having an activity of 8.8 nmol/min/
mg protein. Whether the single low value reflects a
slow acetylator and polymorphism, or the lower
end of the normal range, is not known.
The metabolism of the cysteine conjugate of
HCBD by N-acetyltransferases occurred at sig-
nificantly faster rates than that by b-lyases in both
rat and human kidney thus minimising the amount
metabolised to toxic intermediates. In human
kidney, the rate of metabolism of the N-acetylcys-
teine conjugate back to the cysteine conjugate by
acylases is low, below the limit of detection,
further minimising the amount of cysteine con-
jugate available for metabolism by b-lyases. In
contrast, in the rat acylase activity was high thus
increasing the amount of cysteine conjugate avail-
able. Using these experimentally measured meta-
bolic rate constants in a PB-TK model has enabled
the uptake and metabolism of HCBD to be
compared in rats and humans and the potential
toxicity of HCBD to humans to be predicted from
the known toxicity in the rat. Following exposure
of rats and humans to atmospheric concentrations
of HCBD, metabolism by the b-lyase pathway is
approximately 20-fold less in humans than in the
rats at the same dose level. This prediction by the
model is consistent with the lower uptake, lower
glutathione S-transferase and b-lyase activities,
and the lack of acylase activity in human kidney.
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