TOXICOLOGY AND APPLIED PHARMACOl OGY 44. 391-399 (1978)

Comparison of the Fate of Vinyl Chloride following Single
And Repeated Exposure in Rats
P. G. Watanabe,' J. A. Zempel, and P. J, Glhring
Toxicology Research Laboratory, Health and Environmental Research, Dow Chemical U.S.A-, Midland,
Michigan 48640
Received July II, 1977; accepted October 19,1977

Comparison of the Fate of Vinyl Chloride following Single and Repeated Exposure in Rats.
Watanabe. P. G., Zempel. J. A., and Gehring, P. J. (1978). Toxicol. Appl, Pharmacol. 44,
391-399. Rats were exposed by inhalation to 5000 ppm of nonlabeled vinyl chloride (VC) 6
hr/day, 5 days/week for 7 weeks. On the last day of repeated exposure “C-labeied VC W3S used.
The fate of the I l4C I VC was compared in a group of rats exposed repeatedly to a group exposed
simultaneously for a single 6 hr period to 5000 ppm of |'*CIVC. The routes and rates of
excretion of UC activity were the same for the two experimental groups. The activity of
microsomal enzymes, as reflected by aniline hydroxylase and p-nitroanisole O-demethylase
derived from 9000 g liver supernatants, was essentially the same in rats exposed once or
repeatedly or in nonexposed control rats. Covalent binding to hepatic macromolecules was
greater in rats repeatedly exposed as compared to those subjected to a single exposure. These
results indicate that repeated exposure to VC does not induce its biotransformation. However,
the increase in hepatic macromolecular binding indicates that repeated exposure augments the
reaction of electrophilic metabolites with macromolecules, and this may be expected to enhance
potential toxicity including carcinogenicity.

While many pharmacokinetic and metabolic studies have been conducted on vinyl
chloride (VC), most of these investigations have concentrated on the fate of VC during
and following single exposure. Since cancer has been induced by long-term repeated
exposure of both experimental animals and man to VC, it is important to consider
alterations in the disposition of VC in the body which may occur after repeated
exposure.
In preliminary studies, monochloroacetic acid was tentatively identified as a major
urinary metabolite following repeated exposure (6 hr/day, 5 days/week for 9 weeks) to
5000 ppm of nonlabeled VC (Hefner et a/., 1975). However, this metabolite has not
been found in rats given a single exposure to |,4C|VC (Watanabe et al., 1976). This
raised the question of whether the biotransformation of VC may be altered upon
repeated exposure.
A change in the biotransformation of VC after repeated exposure is particularly
important in assessing the hazard of exposure to VC since evidence suggests that VC is
metabolized to a reactive metabolite which is ultimately responsible for its toxic
manifestations. This concept is supported by studies showing an enhancement of the
' To whom correspondence should be sent.
39 1

0041 00l!X/7t!'n44: IHVIS02 00/0
Copyright C1 1978 hv Academic Press. Inc
All rights of reproduction m
iorm reserved

Primed m Great Britain

SIj 041647

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392

WATANABE, ZEMPEL, AND GEHR1NG

mutagenic activity of VC to bacteria when microsomal and soluble enzymes arc added
to the system to provide for metabolic transformation (Bartsch el al., 1975: Malavielle
el al., 1975; Rannug el at., 1974). In addition, recent reports demonstrated that liver
microsomal enzymes, in vitro, mediate the binding of VC metabolites to the microsomes
(Kappus et al., 1975), protein sulfhydryl groups, RNA (Bolt et al., 1975), and
adenosine (Barbin et al., 1975). That the biotransformation of VC is involved intimately
in its toxic response was further substantiated when Reynolds et al. (1975a) showed
that acute hepatotoxicity could be produced by VC if the animals were pretreated with
phenobarbital or Arochlor 1254, inducers of microsomal enzyme activity. Reynolds et
al. (1975b) also reported that a single 6-hr exposure to 5% VC deactivates cytochrome
P-450 and other components of the mixed-function oxidase system.
Since there is evidence suggesting that repeated exposure to VC may alter its biotransformation and since the metabolism of VC is associated with its toxicity, the
objective of the current study was to determine whether the fate of VC is indeed altered
with repeated exposure. This was accomplished by exposing rats to nonlabled VC for 7
weeks and, on the last exposure, to 14C-labeled VC. The fate of the (l4C|VC was then
followed for 72 hr and compared to animals receiving a single exposure.

METHODS

Material. Vinyl chloride gas (Matheson Gas Products) of 99.9% minimum purity was
used throughout the study. 14C-Labeled VC was synthesized from I l,2-14C]-dichloroethane (New England Nuclear, Lot No. 819-292, 4.8 mCi/mmol) immediately prior to
use (Wagner and Muelder, 1975). The synthesized [14C]VC has been reported to be 9596% radiochemically pure (Wagner et al., 1975). Nonlabeled VC (Matheson Gas
Products) was mixed with the "14C material to obtain the desired specific activity.
Typically, 40 ml of the [14CIVC-helium gas mixture was injected into a 10-liter Saran
bag (Anspec, Inc.) containing the desired quantity of nonlabeled VC.
Animals. Male Sprague-Dawley rats (Spartan Research Laboratory) with an initial
weight of 160-180 g were used in the study. All animals were housed in rooms in which
constant humidity, temperature, and 12 hr light-dark cycle (8:00 am-8:00 pm) were
maintained. Food and water were provided ad libitum except during the exposure.
Exposures were conducted between 9:00 am and 4:00 pm (EST).
Exposure. All rats were exposed under dynamic conditions to a nominal
concentration of 5000 ppm of VC (treated) or room air (control) in 30-liter glass
inhalation chambers. VC was metered into the chamber air flow (~6 liters/min) with a
dual syringe pump. The nominal concentration of VC was determined from the ratio of
the rate at which the VC gas was dispensed and the total chamber air flow. The
analytical concentration of VC was monitored continuously by recirculating a fraction
of the chamber atmosphere through an infrared spectrophotometer (Wilks) set at 10.6
pm. The rats repeatedly exposed to VC were exposed 6 hr/day, 5 days/week for 7
weeks (32 exposures in 44 days). The mean analytical concentration of VC over the 7
week exposure period was 4775 ± 908 (SD) ppm.
On the final day of exposure the animals were subjected to a 6 hr exposure to l4Clabeled VC generated in the same manner as described above. On this final day of

u*

FATE OF VINYL CHLORIDE IN THE RAT

393

exposure, the chamber atmosphere was also analyzed at hourly intervals by gas
chromatography, and at the same times the MC activity was determined by bubbling 1
ml aliquots of the chamber atmosphere into a scintillation solution containing Concifluor (Mallinckrodt Chemical), 2-methoxyethanol, toluene (6:11:83) (Watanabe et al.,
1976). The radioactivity was determined by counting in a liquid scintillation spectro­
meter. The mean analytical concentration determined on the final exposure day, when
the animals were exposed to 14C-labeled VC, was 4600 ±311 (SD) ppm. The specific
activity was 50 dpm//tg of VC.
The inhalation chamber was operated in a laboratory fume hood to prevent
contamination of the working environment. After transit through the inhalation
chamber the [14C]VC was adsorbed on activated charcoal. These traps were disposed
as radioactive waste according to standard regulations.
Procedure. Eight rats were exposed repeatedly to VC as described previously. On the
last day, five additional unexposed rats and the eight exposed repeatedly were exposed
to 5000 ppm of (14C|VC for 6 hr. Following this final exposure to [l4C|VC, three of the
eight exposed repeatedly and two of the five exposed once were placed in glass Rothtype metabolism cages for the collection of urine, feces, and expired air.
Room air was drawn through the cages at 400-500 ml/min. The exiting air was
passed through a series of traps to collect the expired [14C]VC and 14CO,. The air
leaving the chamber was passed first through a glass tube containing about 40 g of
Drierite (W. A. Hammond Drierite Co.) to remove moisture. Subsequent transit
through a series of two cold finger traps containing 50 ml of toluene, 2-methoxyethanol
(80:20) and a single trap containing 120 ml of 5 m ethanolamine in 2-methoxyethanol
enabled the collection of [14CiVC and 14CO,, respectively. The cold finger traps were
immersed in 2-methoxyethanol-dry ice baths throughout the collection periods. The
trap for CO, was maintained at room temperature.
Samples of excreta were collected for 72 hr after termination of exposure and
analyzed for 14C activity. Expired VC was collected at 0.5-hr intervals for 3 hr; the CO,
trap and urine receptacle (immersed in dry ice bath) were changed at 12-hr intervals for
72 hr; and feces were collected every 24 hr. At the termination of the study (72 hr) the
animals were killed by a blow to the head, and samples of tissues (fat, kidney, liver)
were collected for analysis of ,4C activity. The remaining carcass was skinned and
homogenized (50% w/v) in distilled water and analyzed for 14C activity. The samples of
excreta and tissue were prepared for scintillation counting as described previously
(Watanabe et a/., 1976).
Carbon-14 activity was determined by counting in a Mark II or Mark III liquid
scintillation spectrometer (Searle). External standard-channels ratios were used to deter­
mine the counting efficiency. The counts per minute were converted to disintegrations
per minute using a standard quench curve.
The remaining rats in the groups exposed repeatedly and singly to VC, five and three
respectively, were killed by a blow to the head immediately following exposure. A piece
of liver was used to prepare a 9000 g supernatant in 1.15% KC1 in order to determine
aniline hydroxylase (LaDu el al., 1971) and p-nitroanisole O-demethylase (Kinoshita et
a!., 1966) activity. Macromolecular binding of radioactivity to hepatic tissue was
determined by the method of Jollow et al. (1974). This method measures nonextractable radioactivity from a trichloroacetic acid precipitate which includes protein,

sc

394

WATANABE, ZEMPEL, AND GEHRINC

lipids, and nucleic acids. The carcass was analyzed for total radioactivity as described
above.
A second experiment was conducted to confirm parameters obtained for binding of
radioactivity to hepatic macromolecules. The methodology was the same as described
above. A group of rats was repeatedly exposed to VC 14821 ± 259 (SD) ppml for 8
weeks. On the last day of exposure 1|4C|VC was used and an additional four rats
(singly exposed group) were added. The mean analytical concentration of the l'4C|VC
on the last day of exposure was 5065 + 30 (SD) ppm and the specific activity was 34
dpm/jUgofVC.
RESULTS

Excretion of 14C activity within 72 hr after a single or repeated exposure to 5000 ppm
of [14C1VC is shown in Table 1. The percentage of l4C activity excreted by each route
as well as the total milligram equivalents of VC recovered were essentially identical for
the singly and repeatedly exposed groups. The majority of l4C activity eliminated was
expired as VC perse.
TABLE 1
Percentage

i4C-Activity

Eliminated by Rats During 72 hr Following Inhalation
Exposure to 5000 ppm of 114C)Vinyl Chloride"
Single exposure (2)#

Expired:
As VC
As C02
Urine
Feces
Carcass and tissues
Total VC
recovered

Repeated exposure (Z)h

(%)

(mg equiv of VC)

(%)

(mg equiv of VC)

54.5 + 3.5
8.0 + 1.4
27.1 + 2.1
3.2 + 2.5
7.3 + 2.5

14.00
2.05
6.9.3
0.80
1.89

53.7 + 2.1
9.6 ± 1.6
25.7 + 1.4
1.4 + 0.4
9.7 + 1.6

12.94
2.27
6.21
0.32
2.32

25.67

24.07

" Expressed as percentage of the total l4C activity recovered, mean + SD.
b Number of rats.

The time course for expiration of [ l4C]VC per se (Fig. 1) and urinary excretion of 14C
activity (Fig. 2) were also essentially identical for the singly and repeatedly exposed
rats. The curves were fit by linear regression analysis of the logarithmically transformed
data. The estimate of the apparent first-order rate constant for expiration of VC was
0.023 ± 0.01 (SD) min"1 which corresponds to a half-life of 30 min. The elimination of
urinary l4C activity was biphasic. An estimate of the apparent first-order rate constant
for the initial portion of the urinary excretion curve from 12 to 36 hr was 0.155 ± 0.02
(SD) hr-1 which corresponds to a half-life of 4.47 hr. The data for the slow phase of
urinary excretion were extremely variable: therefore no attempt was made to estimate
the excretion rate. Less than 1% of the radioactivity excreted in the urine occurred
during the slow phase.

i

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FATE OF VINYL CHLORIDE IN THE RA'l

395

Urinary 14C activity was separated by thin-layer chromatography in n-butanol,
acetone, H20 (50:20:30) on cellulose and in n-butanol, acetic acid, H20 (80:20:20) on
silica gel. The profiles of radioactivity for rats exposed repeatedly or singly were
qualitatively similar and no significant radioactivity was associated with the fly value of
a standard of monochloroacetic acid.
The concentration of radioactivity detected in tissue 72 hr after exposure revealed no
statistically significant difference between rats exposed once or repeatedly to VC (Table

Time, Hours

Fig. I. Expired vinyl chloride (milligrams) versus time following a 6 hr exposure to 5000 ppm of

I MC|VC. Repeatedly exposed rats (▲) and singly exposed rats (•).

2). It does appear that, in those exposed repeatedly, more radioactivity may have been
retained in the liver and skin; however, the number of animals used does not provide for
an adequate statistical evaluation.
The effect of VC on xenobiotic drug metabolism by liver 9000 g supernatants, as
reflected by aniline hydroxylase and p-nitroanisole O-demethylase activity, is presented
in Table 3. Neither single nor repeated exposure to 5000 ppm of VC altered discernibly
the enzyme activity in either system when compared to air-exposed controls.
The total amount of VC biotransformed and the hepatic macromolecular binding of
l4C activity following single and repeated exposure are shown in Table 4. The total
amount of VC biotransformed was not significantly different between the two groups.
However the hepatic macromolecular binding in the first experiment appeared to be
increased in the rats exposed repeatedly. When the protein binding was corrected for the

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396

WATANABE, ZEMPF.L. ANB GEHRING

Fig. 2. ,4C-Activity excreted in the urine expressed as percentage of the recovered radioactivity versus
time following a 6 hr exposure to 5000 ppm of [l4C|VC. Repeatedly exposed rats (A) and singly exposed
rats (•).

amount of VC biotransformed (B/A x 100) a statistically significant increase was
found. Because the increased binding in repeatedly exposed rats was not definitive in the
first experiment, the experiment was repeated and the results are shown in the lower
portion of Table 4. The second study confirmed the observation that rats repeatedly
TABLE 2
Percentage “C-Activity 72 hr Following Inhalation
Exposure of Rats to 5000 ppm of Vinyl Chloride"
l4C Activity/g of tissue (%)
Tissue

Single exposure (3)*

Repeated exposure (2)*

Liver
Kidney
Fat
Skin
Carcass

0.119 + 0.022
0.062 + 0.026
ND"
0.046 + 0.015
0.030 + 0.014

0.157 + 0.028
0.070 + 0.006
NDC
0.080 + 0.019
0.039 +0.011

" Expressed as percentage of the total ,4C activity metabolized,
mean + SD.
4 Number of rats per group.
‘ Not detectable, detection limit of 3 tig of VC equiv/g of fat or
0.03% l4C activity metabolized/g of tissue.

SV

0

\

F A II

01

\ IN VI

till < > K1 I ) I

IN

I III

347

KM

exposed to VC hind about 20--25"n more reacme metabolite to hepatic macromoleeules
than do rats exposed once. It is riot clear win the magnitude of'the binding was slightlv
greater in the second experiment. Nonetheless, these results indicate that a huger
fraction of the biotransformcd VC reacts coxalently with hepatic macromoleeules in
rats exposed repeatedly.

TAB! t
Ffflci

of

Vinyl Chloridi

on

3

Dkig Mt tabolism By

a

4000 g

SlJPF.RNAl AN T FRXCIION OF LlVF'R OF RaTS"

Amount lug of producing of liver/hr)

Control MV1
Single VC exposure (3V1
Repeated VC exposure (5)h

■\mlme
hydroxylase

p Nilroanisole
O-demethvlase

65 - 16
71 - 7
S3 - 10

226 + 22
254 + 45
217 + 31

11 Animals were killed immediately following the last exposure and enzyme
activity was assayed Values are mean * SF>
p Number of rats per group.

TABl.f 4
Toi al Mfiabolism and Hfpatic Macromolf.cdlar Binding Followinc, SiM.it
Rlpkated F.xpqsl'RF. of Rats to 5000 ppm of Vinyl Chloride"
A
VC equivalents
metabolized

Binding corrected
for metabolism

lag)

B
VC equivalents
hound
(ug/g of protein)

Single exposure
Repeated exposure

9265 + 1467
8718 + 895

114+10
124 t 10

1.12+0.13
1.43 +0.16'

Experiment was repeated
Single exposure
Repeated exposure

8746 + 882
9421 + 482

148 + 25
195 + 24‘

1.64 + 0.28
2.07 + 0.25'

or

(B/A v I00)'1

" Values are ihe mean *■ SD (three to fixe rats per group)
h The ratio of B A ■ 100 was calculated from individual animal data.
1 Statistically significantly different from the single exposure. Student / test (p < 0.05),

DISCUSSION
Repeated exposure of rats to 5000 ppm of I l4C I VC did not alter discernible the
routes or rates of excretion of radioactivity or qualitatively the excretory products
formed from

VC or VC per se. These results do not substantiate the previous

preliminary observation that monochloroacetate may be a major biotransformation
product of VC (Hefner ei ai. 1975).

sv

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398

WATANABE, 7.EMPEL, AND GLHRENG

A most significant finding in the study was a significantly increased amount of radio
activity bound covalently to macromolecules of rats exposed repeatedly to VC versus
those exposed once. An associated observation was the retention of an apparently
greater level of radioactivity in the liver of repeatedly exposed rats 72 hr after exposure
than those exposed once. These results indicate that toxic manifestations, including
carcinogenicity, associated with the reaction of reactive metabolites of VC with macro­
molecules may be enhanced by repeated exposure to VC.
While the binding of reactive metabolites of VC to hepatic macromolecules was
enhanced following repeated exposure, no differences were observed in the activity of
hepatic microsomal enzymes to the substrates aniline or p-nitroanisole in any of the
treatment groups when compared to nonexposed control rats. Thus, it did not appear
that exposure to VC at this concentration influenced microsomal metabolism. However,
in contrast to these findings was the observation by Reynolds et al. (1975b) that the
cytochrome P-450 content and the oxidative TV-demethylation of aminoantipyrine and
ethylmorphine were markedly depressed in rats following exposure to 50,000 ppm of
VC for 6 hr. The difference between our study and that of Reynolds et al. (1975b) is
that we used substrates which cause a “type II” binding spectra compared to their use of
substrates producing a “type I” binding spectra with hepatic microsomes. In addition
Reynolds et al, (1975b) used a 10-fold greater concentration of VC. The apparent
discrepancy can be explained by the recent observation that VC causes a “type I"
binding spectra when incubated with hepatic microsomes (Salmon, 1976; Ivanetich et
al., 1977), and furthermore it has been demonstrated (Ivanetich et al.. 1977) that high
concentrations of VC can degrade cytochrome P-450. Thus it appears that VC is
metabolized by the hepatic microsomal enzymes and is capable of inhibiting
metabolism of other substrates by competitive inhibition or by degradation of cyto­
chrome P-450.
In conclusion, the results of these studies showed that repeated exposure to high
levels of VC did not alter discernibly the routes or rates of excretion of radioactivity
when compared to rats subjected to a single 6 hr exposure to 5000 ppm of [ 14C1 VC. Of
particular significance was evidence that the binding of reactive metabolites of VC with
hepatic macromolecules was enhanced by repeated exposure to high levels of VC.
Associated with this may be expected an enhanced toxicity, including carcinogenicity.
The reason for the enhanced covalent binding with repeated exposure is under
investigation.
ACKNOWLEDGMENT
The authors wish to express their appreciation to M. M. Schlachter for technical assistance.

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FATE OF V1N Yt. CMLORIDF IN TFIF R A I

399

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