!

^ tr

FATE OF VINYL CHLORIDE IN THE RAT

exposure. me cnamber atmosphere was also analyzed at hourly intervals by gas
cnromatograDny. and at the same times the 14C activity was determined by bubbling 1
mi aiiauois of the chamber atmosphere into a scintillation solution containing Concifiuor (Mallincxrodt Chemical). 2-methoxyethanol, toluene (6:11:83) (Watanabe ei 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 ‘4C-labelea VC, was 4600 r 311 (SD) ppm. The specific
activity was 50 dpm/ug 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 (MC]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 ana the eight exposed repeatedly were exposed
to 5000 ppm of! UC!VC for 6 hr. Following this final exposure to [lJC|VC, three of the
eight exposed repeatedly and two of the five exposed once were placed in glass Rothtype metaoonsm 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 traos 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
Driente (W. A. Hammond Drierite Co.) to remove moisture. Subsequent transit
through a series of two cold finger traps containing 50 ml of toluene, 2-methoxyethanoi
(S0:20) and a single trap containing 120 ml of 5 M ethanolamine 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 UC activity. Expired VC was collected at 0.5-hr intervals for 3 hr; the CO,
trao 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 l4C activity. The remaining carcass was skinned and
homogenized (50% w/v) in distilled water and analyzed for WC activity. The samples of
excrcia and tissue were prepared for scintillation counting as described previously
(Waianabe el al.. 1976).
Carbon-U activity was determined by counting in a Mark II or Mark III liquid
scintillation spectrometer (Searie). 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 io the head immediately following exposure. A piece
of h\er was tisea to nrenare a 9000 g supernatant in 1.15% K.C1 in order to determine
an.line hydroxy uisc (LaOu ct
1971) and p-nitroanisole 0-demethyiasc (Kinoshiia ei
ui.. 1966) activity. Macromoiecular binding of radioactivity to hepatic tissue was
determined by me method of Jollow ct <t/. (1974). This method measures nonextraetable radioactivity from a trichloroacetic acid precipitate which includes protein.

R&S162565

v-

393

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
raoioactivitv to hepatic macromoiecuies. The methodology was the same as described
above. A group of rats was repeatedly exposed to VC [4821 r 259 (SDl ppmi for 3
weeks. On the last day of exposure iMC!VC was used and an additional four rats
f singiy exposed group; were added. The mean analytical concentration of the ;'JC!VC
on the last day of exposure was 5065 z 30 (SD) ppm and the specific activity was 34
dpm/.ug of VC.
RESULTS

Excretion of 14C activity within 72 hr after a single or repeated exposure to 5000 Dpm
of 1UC|VC is shown in Table 1. The percentage of IJC 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 IJC activity eliminated was
expired as VC perse.
TABLE I
Percentage uC-Acttvity Eliminated by Rats During 72 hr Following Inhalation
Exposure to 5000 ppm of 1uC!Vinyl Chloride1'
Single exposure (2)°

Expired:
As VC
As CO,
Urine
Feces
Carcass and tissues
Total VC
recovered

Repeated exposure (3)"

(%)

(mg equiv of VC)

(°o)

(mg equiv of VC)

54 5 * 3 5

14.00
2.05
6.93
0.80
1.S9

53.7 z 2.1
9.6 - 1.6
25.7 - 1.4
1.4 0.4
9.7 - 1.6

12.94
2,27

8.0 r 1.4
27.1 - 2.1
3.2 - 2.5
7.3 - 2.5

25.67

6.21

0.32
7.
24.07

J Exoresied as percentage of ihe total IJC activity recovered, mean t SD.
* Number of rats.

The time course for expiration of i IJC!VC per se (Fig. 1) and urinary excretion of UC
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 z 0.01 (SD) min-1 which corresnonds 10 a half-life of 50 min. The elimination of
urinarv MC activity was hiphasic. An estimate of the apparent first-order rate constant
for the initial portion of the urinary excretion curve from 12 to 56 hr was 0.155 r 0.02
iSD) hr -1 which corresnonds ;o a hail'life of 4.47 hr. The data for the slow pnase of
,
urinarv excretion were extremely variame: therelorc no attempt was made to estimate
[he excretion rate. Less than I"., of the radioactivity excreted in the urine occurred
during ihe siow phase.

jj

Qo
c/j

FATE OF VINYL CHLORIDE IN THE RAT

395

Urinary l4C activity was separated by thin-layer chromatography in n-butanoi.
acetone. H.O (50:20:30) on cellulose and in n-butanol. acetic acid. H.O (80:20:20) on
silica gel. The profiles of radioactivity for rats exposed repeatedly or singiv were
qualitatively similar and no significant radioactivity was associated with the R{ 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

T.m*, How

Fig. I. Expired vinyl chloride (milligrams) versus time following a 6 hr exposure to 5000 ppm of
1 “ClVC. Repeatedly exposed rats 1 Ai and singly exposed rats 1 •).

R&S162567

2). It docs 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 xcnobiotic drug metabolism by liver 9000 g supernatants, as
reflected by aniline hvdroxyiase and p-nuroamsole O-demethylase activity, is presented
in Table 5. Neither single nor repeated exposure to 5000 ppm of VC altered discernible
the enzyme activity in either system when compared to air-exposed controls.
The total amount oi VC hioiransformcd and the hepatic macromolecular binding ot
MC activity following single and repeated exposure arc shown in Table 4. The total
amount of VC biotransformea was not sisnmcantlv different between the two groups.
However the hepatic macromolecular binding in the first experiment appeared to be
increased in the rats exposed rcpcatedlv. When the protein binding was corrected for the

«

396

watanabe. zempel. and gehring

0

U

:4

JO

60

77

0*

7.m* Hours
F*ig. 2. “C-Aeuvity excreted in the urine expressed as percentage of the recovered radioactivity versus
time following a ft nr exposure to 5000 ppm of i'‘C!VC. Repeatedly exposed rats iai 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 '2 hr Following InhalationExposure of Rats to 5000 npm of Vinyl ChlorideUC Activity g of tissue <°i'i)
Tissue

Single exposure <3

Repeated exposure (2)'

Liver

0.119 * 0.022

Kidney

0.0A2 - 0.026

0.157 - 0.02S
0.070 - 0.006

Fat
Skin
Carcass

S'D‘
0.046 - 0.015
0.030 - 0.014

0.0S0 - 0.019
0.039 - 0.011

as pcrccnl.iee ot ;he (olat

l4C activity mctaoolieed.

:fle:in * SD.
* Numncroi rats per "rouf>.
NtM detectable, Jetceuon limit
OOV’-i ,jr activity metabolized e oi

3 j*

\*C

oquiws

»m" fat »>r

R&S162568

‘ Rxprc'.scd

NDf

i

FATE OF VINYL CHLORIDE IN THE RAT

397

exposed to VC bind about 20-25°h 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 exoenment. Nonetheless, these results indicate that a larger
fraction of the biotransformed VC reacts covalently with hepatic macromolecules in
rats exoosed repeatedly.
TABLE 3
Effect

of

Vinyl Chloride on Drug Metabolism By a 9000 g
Supernatant Fraction of Liver of Rats"
Amount (fiz of product/g of liver/hr)

Control (4i®
Sinsle VC exposure (31®
Repeated VC exposure (5)®

Aniline
hydroxylase

p-Nitroamsole
O-demethylase

65 z 16
71-7
83 z 10

226 - 22
254 - 45
217 z 31

* Amman were killed immediately following ihe last exposure and enzyme
activity was assavec. Values are mean - SD.

* Numner of rats ocr group.
TABLE 4

Total Metabolism and Hepatic Macromolecular Binding Following Single
Repeated Exposure of Rats to 5000 ppm of Vinyl Chloride*'
Binding corrected
for metabolism

A
VC eauivaients
metabolized
( Ug)

VC equivalents
bound
(p%!% of protein)

Single exposure
Repeated exposure

9265 z 1467
S718 z 895

114 ± 10
124 t 10

1.12 z 0.13
1.43 z 0-16-

Experiment was repeated
Single exposure
Repeated exposure

8746 = 882
9421 z 482

148 t 25
195 Z 24*

1.69 z 0.28
2.07 z 0.25*

8

or

(B/A \ 100)®

J Values arc the mean - SD 'three to rive rats Der croup).
"The ratio of B/A ■ 100 was calculated from individual animal data.
r Statisticallv vignineantlv different from the sinclc exposure. Student t test (p < 0.051.

Repealed exposure of rats to 5000 ppm of |IJCIVC did not alter diseermbly the
routes or rates of excretion of radioactivity or qualitatively the excretory products
formed from VC or VC per ?e. These results do not substantiate the previous
preliminary* observation that monochloroacctate may be a major biotransformation
product of VC i Heine: er .ii.. 1975).

R&S162569

DISCUSSION

(

398

watanabe. zempel. and gehring

A most significant finding in the study was a significantly increased amount of radio­
activity bound covalently to macromoiecuies 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 resuits indicate that toxic manifestations, .including
carcinogenicity, associated with the reaction of reactive metabolites of VC with macro­
moiecuies may be enhanced by repeated exposure to VC.
While the binding of reactive metaoolites of VC to hepatic macromolecules was
enhanced following repeated exposure, no differences were observed in the activity of
heoatic microsomal enzymes to the substrates aniline or p-nitroanisolc in any of the
treatment grouDS 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
cttochrome /M50 content and the oxidative .V-demethyiation of aminoantipvrine and
etnyimorpnine 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 11“ 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 /M50. 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
leveis of VC did not alter discermbiy the routes or rates of excretion of radioactivity
when compared to rats subjected to a single 6 hr exposure to 5000 ppm of l1JC|VC. Of
particular significance was evidence that the binding of reactive metabolites of VC with
hepatic macromoiecuies 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 covaieni binding with repeated exposure is under
investigation.
ACKNOWLEDGMENT
The authors wish lo express iheir appreciation to M. M. Seltlachter lor technical assistance.

references

-iCv—15~.

R&S162570

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