__ <-nn-c*>s')
R&S 136339

TOXICOLOGY AND APPLIED PHARMACOLOGY 44, 391-399 (1978)

Comparison of the Fate of Vinyl Chloride following Single
And Repeated Exposure in Rats
P. G. Watanabe,1 J. A. Zempel, and P. J. Gehring
Toxicology Research Laboratory, Health and Environmental Research, Dow Chemical U.SA., 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 14C-labeled VC was used.
The fate of the 1,4C 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 [l4C]VC. The routes and rates of
excretion of 14C activity were the same for the two experimental groups. The activity of
microsomal enzymes, as reflected by aniline hydroxylase and /vnitroanisole 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 el al„ 1975). However, this metabolite has not
been found in rats given a single exposure to [’■‘CIVC (Watanabe el 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
1 To whom correspondence should be sent.
391

0O4l-00RX'78/0442-O39l S02.00/0
Copyright
1978 b> Academic Press. Inc
All rights of reproduction in any form reserved.
Printed in Great Britain

392

30
CO
w
CT>
CO

4*
O

WATANABE, ZEMPEL, AND GEHRING

mutagenic activity of VC to bacteria when microsomal and soluble enzymes are added
to the system to provide for metabolic transformation (Bartsch et al., 1975: Malavielle
et al., 1975; Rannug et al., 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 acuvity. 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 I4C-labeled VC. The fate of the [UC]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. uC-Labeled VC was synthesized from [ I,2-14C]-dichloroethane (New England Nuclear, Lot No. 819-292, 4.8 mCi/mmol) immediately prior to
use (Wagner and Muelder, 1975). The synthesized [l4C]VC has been reported to be 95—
96% radiochemically pure (Wagner et al., 1975). Nonlabeled VC (Matheson Gas
Products) was mixed with the I4C material to obtain the desired specific activity.
Typically, 40 ml of the [14C|VC-helium gas mixture was injected into a 10-liter Saran
bag (Anspec, Inc.) containing the desired quantity of nonlabeled VC.
Animats. 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 condidons 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 concentradon of VC was determined from the ratio of
the rate at which the VC gas was dispensed and the total chamber air flow. The
analyucal 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 m'ean 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

FATE OF VINYL CHLORIDE IN THE RAT

393

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 et ai. 1971) and p-nitroanisole O-demethylase (Kinoshita et
al.. 1966) activity. Macromolecular binding of radioactivity to hepatic tissue was
determined by the method of follow et al. (1974). This method measures nonextractable radioactivity from a trichloroacetic acid precipitate which includes protein,

R&S 136341

exposure, the chamber atmosphere was also analyzed at hourly intervals by gas
chromatography, and at the same times the UC 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 ai.
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/tig 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 [14C]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 [I4C]VC and l4CO,. 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 ethanolaminc in 2-methoxyethanol
enabled the collection of [,4C]VC 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 14C 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 ai, 1976).

394

WATANABE, ZEMPEL. AND GEHRING

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 [4821 ± 259 (SD) ppml for 8
weeks. On the last day of exposure [UC]VC was used and an additional four rats
(singly exposed group) were added. The mean analytical concentration of the [14C]VC
on the last day of exposure was 5065 ± 30 (SD) ppm and the specific activity was 34
dpm//ig of VC.
RESULTS
Excretion of ,4C activity within 72 hr after a single or repeated exposure to 5000 ppm
of (14C]VC is shown in Table 1. The percentage of 14C 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 14C activity eliminated was
expired as VC perse.
TABLE 1
Percentage ‘‘C-Activity Eliminated by Rats During 72 hr Following Inhalation
Exposure to 5000 ppm of ['■'CIVinyl Chloride11
Single exposure (2)4

R&S 136342

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

Repeated exposure (3)4

(%)

(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
6.93
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 14C activity recovered, mean ± SD.
4 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 ,4C 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~* 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.

FATE OF VINYL CHLORIDE IN THE RAT

395

Urinary l4C 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 Rf 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

R&S 136343
Fig. 1, Expired vinyl chloride (milligrams) versus time following a 6 hr exposure to 5000 ppm of
[I4C1VC. Repeatedly exposed rats (A) 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
14C 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

396

WATANABE, ZEMPEL, AND GEHRING

R&S 136344

Fig. 2. MC-Activity excreted in the urine expressed as percentage of the recovered radioactivity versus
time following a 6 hr exposure to 5000 ppm of l,4C|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 14C-AcnvrrY 72 hr Following Inhalation
Exposure of Rats to 5000 ppm of Vinyl Chloride"
14C Activity/g of tissue (%)

■a
'

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

0 Expressed as percentage of the total IJC activity metabolized,
mean + SD.
4 Number of rats per group.
c Not detectable, detection limit of 3 MZ of VC equiv/g of fat or
0.03% MC activity metabolized/g of tissue.

397

FATE OF VINYL CHLORIDE IN THE RAT

exposed to VC bind about 20-25% more reactive metabolite to hepatic macromolecules
than do rats exposed once. It is not clear why the magnitude of the binding was slightly
greater in the second experiment. Nonetheless, these results indicate that a larger
fraction of the biotransformed VC reacts covalently with hepatic macromolecules in
rats exposed repeatedly.
TABLE 3

Effect of Vinyl Chloride on Drug Metabolism By a 9000 g
Supernatant Fraction of Liver of Rats4
Amount (pg of product/g of liver/hr)

Control (4)*
Single VC exposure (3)*
Repeated VC exposure (5/

Aniline
hydroxylase

p-Nitroanisole
O-demethylase

65 ± 16
71 ± 7
83 + 10

226 ± 22
254 + 45
217 + 31

4 Animals Were killed immediately following the last exposure and enzyme
activity was assayed. Values are mean + SD.
4 Number of rats per group.

TABLE 4

Total Metabolism and Hepatic Macromolecular Binding Following Single
Repeated Exposure of Rats to 5000 ppm of Vinyl Chloride4
A
VC equivalents
metabolized

Binding corrected
for metabolism

(PS)

B
VC equivalents
bound
(pg/g of protein)

Single exposure
Repeated exposure

9265 + 1467
8718 ± 895

114+10
124 + 10

1.12 ±0.13
1.43 ± 0.16f

Experiment was repeated
Single exposure
Repeated exposure

8746 ± 882
9421 ± 482

148 + 25
195 + 244

1.69 + 0.28
2.07 ± 0.25c

or

(B/A x 100)4

° Values are the mean + SD (three to five rats per group).
4 The ratio of B/A x 100 was calculated from individual animal data.
‘ Statistically significantly different from the single exposure. Student t test (p < 0.05).

Repeated exposure of rats to 5000 ppm of [14ClVC did not alter discemibly 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 et al, 1975).

R&S 136345

DISCUSSION

37
ffo
W
C4
O)
CO

-b

o>

398

WATANABE. ZEMPEL. AND GEHRINO

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 macromolecules 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 (V-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 additiqp
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 discemibly the routes or rates of excretion of radioactivity
when compared to rats subjected to a single 6 hr exposure to 5000 ppm of [UC]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.

REFERENCES

Barbin, A., Bresil. H., Croisy, A., Jacquignon. P., Malavielle. C.. Montesano. R., and
Bartsch, H. (1975). Liver microsome mediated formation of alkylating agents from vinyl
bromide and vinyl chloride. Biochem. Biophys. Res. Commun. 67, 596-603.

Bartsch. H., Malavielle, C., and Montesano. R. (1975). Human, rat. and mouse liver
mediated mutagenicity of vinyl chloride in Salmonella typhimurium strains. Ini. J. Cancer 15,
429—437.

FATE OF VINYL CHLORIDE IN THE RAT

399

Bolt. H. M., Kappus, H., Butcher. A., and Bolt. W. (1975). Metabolism of vinyl chloride.
Lancet, 1. 1425.

Hefner. R. E., Jr.. Watanabe. P. G., and Gehring. P. J. (1975). Preliminary studies of the
fate of inhaled vinyl choride monomer (VCM) in rats. Ann. N. Y. Acad. Sci. 246, 135-148.
Ivanetich, K. M., Aronson, I., and Katz, I. D. (1977). The interaction of vinyl chloride with
rat hepatic microsomal cytochrome P-450 in vitro. Biochem. Biophvs. Res. Commun. 74,
1411-1418.

Jollow, D. J., Thorgeirsson. S. S., Potter. W, Z., Hashimoto. M.. and Mitchell, J. R.
(1974). Acetaminophen-induced hepatic necrosis VI. Pharmacology 1Z 251-271.

Kappus, H., Bolt, H. M., Butcher, A., and Bolt. W. (1975). Rat liver microsomes catalyze
covalent binding of '■‘C-vinyl chloride to macromolecules. Nature (London) 257, 134-135.

Kinoshita, F. K., Frawley, J. P., and DuBois, K. P. (1966). Quantitative measurement of
induction of hepatic microsomal enzymes by various dietary levels of DDT and toxaphene in
rats. Toxicol. Appl. Pharmacol. 9, 505-513.
LaDu, B. N., Mandel, H. G., and Way, E. L. (eds.) (1971). Fundamentals of Drug
Metabolism and Drug Disposition, pp. 566-569. The Williams and Wilkins Co., Baltimore,
Md.

Malavielle. C„ Bartsch. H., Barbin. A., Camus, A. M., and Montesano. R. (1975).
Mutagenicity of vinyl chloride, chloroethyleneoxide, chloroacetaldehyde and chloroethanol.

Biochem. Biophvs. Res. Commun. 63, 363-370.

Rannug, U.. Johansson, A„ Ramel. C., and Washtmeister. C. A, (1974). The mutagenicity
of vinyl chloride after metabolic activation. Ambio 3, 194-197.

Reynolds, E. S., Moslen, M. T., Szabo. S., Jaeger, R. J., and Murphy, S.D. (1975a).
Hepatotoxicity of vinyl chloride and 1,1-dichloroethylene. Amer.J. Pathol. 81, 219—231..

Reynolds, E. S.. Moslen, M. T„ Szabo, S., and Jaeger, R. J. (1975b). Vinyl chloride induced
deactivation of cytochrome P-450 and other components of the liver mixed function oxidase
system: An in vivo study. Res. Commun. Chem. Pathol. Pharmacol. 1Z 685-693.
Salmon. A, G. (1976). Cytochrome P-450 and the metabolism of vinyl chloride. Cancer Lett. Z
109-117.
Wagner, E. R„ and Muelder. W. W. (1975). A procedure for preparing '■'C-labelled vinyl
chloride. Ann. N.Y. Acad. Sci. 246, 152-153.
Wagner, E. R., Muelder. W. W., Watanabe, P. G., Hefner. R. E., Jr., Braun, W. H., and
Gehring, P. J. (1975). Gas chromatographic method for the preparation of MC-labeled vinyl
chloride. J. Label. Compounds 11, 535-542.
Watanabe, P. G., McGowan, G. R., Madrid, E. O., and Gehring. P. J. (1976). Fate of l4Cvinyl chloride following inhalation exposure in rats. Toxicol. Appl. Pharmacol. 37,49—59.

00
So
CO