Toxicology, 14 (1979) 153--166 © Elsevier/North-Holland Scientific Publishers Ltd.
E V A L U A T I O N OF T E R A T O G E N I C I T Y AND B E H A V I O R A L T O X ICIT Y WITH I N H A L A T I O N EXPOSURE O F M A T E R N A L RATS TO TRICHLOROETHYLENE*
MARK A. DORFMUELLER, STEPHEN P. HENNE, RAYMOND G. YORK, ROBERT L. BORNSCHEIN and JEANNE M. MANSON Department of Environmental Health, University of Cincinnati, Cincinnati, OH (U.S.A.) (Received July 25th, 1979) (Accepted October l l t h, 1979)
SUMMARY Female rats were exposed by inhalation to t ri chl oroet hyl ene (TCE) vapors at a c o n c e n t r a t i o n of 1800 -+ 200 p p m t o det erm i ne w h e t h e r exposure before mating and during pregnancy is m o r e detrimental t o reproductive o u t c o m e than exposure either before mating alone or during pregnancy alone. F o u r t r e a t m e n t groups were utilized in a two by t w o factorial design: exposure t o TCE for 2 weeks before mating and during the first 20 days o f pregnancy; TCE before mating and filtered air during pregnancy; filtered air before mating and TCE during pregnancy; and filtered air before and during pregnancy. Significant elevations in skeletal and soft tissue anomalies, indicative o f developmental delay in m at ur a t i on rather than teratogenesis, were observed in the group exposed during pregnancy alone. The mixed funct i on oxidase enzymes, e t h o x y c o u m a r i n and e t h o x y r e s o r u f i n , indicative o f cytoch r o me P-450 and P-448 activities, respectively, were measured in maternal and fetal livers, as well as livers of non-pregnant females, and showed variable levels o f activity n o t u n i f o r m l y related t o t r e a t m e n t or pregnancy. Behavioral evaluation o f offspring indicated a lack of t r e a t m e n t effect in tests o f general activity levels at 10, 20 and 100 days o f age. However, a reduct i on in postnatal b o d y weights was seen in offspring from m ot hers with pregestational exposure. No results indicative o f treatment-related maternal toxicity, e m b r y o t o x i c i t y , severe teratogenicity or significant behavioral deficits were obtained in any o f the t r e a t m e n t groups. *Supported by NIEHS grants No. ES 01601 and No. 3-P30-ES00159 Center for the Study of the Human Environment. Address Correspondence to: Dr. Jeanne M. Manson, Department of Environmental Health, University of Cincinnati, 3223 Eden Avenue, Cincinnati, OH 45267, U.S.A. Abbreviations: MFO, mixed function oxidase; TCE, trichloroethylene.
153
INTRODUCTION Trichloroethylene (TCE) is an unsaturated chlorinated hydrocarbon (molecular formula CI:C=CHC1) widely used in industrial and commercial products. Over 9(Wo of the TCE produced in the United States is utilized as a liquid or vapor cleaning solvent in industrial, automotive, aircraft and aerospace degreasing operations. The remaining percentage is used as a component in many common consumer products including cleansers for automobiles, buffing solutions, spot removers, rug cleaners, pesticides and paints [1,2]. The current Federal occupational exposure limit to TCE is 100 p p m determined as a time-weighted average for an 8-h exposure period with a ceiling limit of 200 p p m [3]. There has been no exact determination of the amount of TCE that is fatal to humans. However, there are numerous case reports of workers w h o have died as a result of TCE exposure. In the best documented cases [4,5] exposUre levels were estimated to be between 1700 and 3300 ppm. Prior to death, the workmen complained of nausea, drowsiness, dizziness, and vomiting. Several investigators [6,7] have reported hepatic necrosis and nephropathy resulting from inhalation of TCE contained in aerosol products. Chronic exposure to TCE has been reported t o result in elevated levels of serum transaminases indicative of damage to the liver parenchyma [8]. However, Milby [9] f o u n d no evidence of abnormal liver or kidney function (SGOT, SGPT, erythrocyte sedimentation rate) in a paint stripping machine operator exposed to 280 ppm TCE. Several studies have reported behavioral alterations in workers exposed to TCE. Stopps and McLaughlin [10] found a progressive decline in psychom o t o r performance with increasing TCE exposure over a range of 100--500 ppm. Similarly, Salvini et al. [11] noted a decrease in performance on a manual dexterity test for subjects exposed to 110 ppm of TCE for t w o 4-h sessions. They also found significant decreases in performance on a perception test, the Wechsler m e m o r y scale and a complex reaction time test. Other reported behavioral changes following acute exposure to TCE include diplopia [12,13], paranoid psychosis and hallucinations [14]. Chronic exposure to TCE has resulted in CNS disturbances [15], loss of tactile sense [16] and in one case, total blindness [17]. TCE was one o f several solvents tested for teratogenic potential by Schwetz et al. [18]. Rats and mice were exposed b y inhalation for 7 h/day on days 6--15 of gestation to 300 p p m of TCE. Dams were sacrificed at term, and evaluation of material toxicity, e m b r y o t o x i c i t y and teratogenicity yielded negative results. The short-term exposure period utilized in this study constitutes the conventional test of teratogenicity which m a y n o t be adequate to predict the teratogenic potential of TCE. Kimmerle and Eben [19] demonstrated that the pharmacokinetic behavior of TCE after acute exposure differs from that after subacute exposure. These investigators found that repeated exposure resulted in continuous elevation of the toxic metabolite, trichloroethanol, in the blood while levels of the parent c o m p o u n d 154
remained relatively stable. Short~term exposure from days 6--15 of gestation could lead to an underestimation of the teratogenicity of TCE insofar as metabolic activation systems generating toxic metabolites may not be induced during the organogenesis period when the conceptus is most susceptible to teratogenic insult. Thus, one aim of the present study was to assess the reproductive consequences of exposure to TCE both before mating and during pregnancy compared to before mating alone or during pregnancy alone. The currently accepted protocol for teratology testing (i.e., sacrifice of pregnant dams at term following exposure during the organogenesis period, and examination o f maternal and fetal tissue) is appropriate for investigation o f structural anomalies in term fetuses, but not of the functional integrity of the offspring. In the latter case, postnatal behavioral evaluation m a y be a more sensitive index of overall functional capacity. As noted previously several studies have found behavioral alterations as a result of chronic or acute exposure to TCE in adult humans. However, systematic investigation o f the behavioral effects in postnatal animals following prenatal exposure to TCE have y e t to be conducted. Thus, a second aim o f the present study was to assess the effects of maternal exposure to TCE prior to and/or during pregnancy on the behavioral development and function of the offspring. METHODS Animals and exposure conditions
One hundred and t w e n t y virgin female Long-Evans hooded rats were purchased from Charles River Breeding Laboratories. Animals were ear-notched for individual identification and toe-clipped for treatment group identification, and quarantined for 2 weeks after arrival. They were singly housed in stainless steel wire b o t t o m cages in a temperature (23°C) and light-controlled r o o m (12:12 light--clark cycle, light commencing at 7 a.m.). Standard laboratory rat chow (Purina 5001) and distilled water were available ad lib. except during residence in exposure chambers. At the beginning of the experiment, female rats weighing approx. 210 g were randomly assigned to 1 of 4 treatment groups, with 30 rats per group as follows: A (++) - TCE exposure before mating and during pregnancy; B ( + - ) -- TCE exposure before mating, filtered air during pregnancy; C ( - +) -- Filtered air before mating, TCE during pregnancy; D ( - - ) -- Filtered air before mating and during pregnancy; The pre-mating exposure was conducted for 6 h a day, 5 days a week for 2 weeks. Following the last day of exposure, the mating regimen began. Two estrous females were placed with 1 breeder male of the same strain overnight.Vaginal smears were conducted the following morning prior to exposure. Day 1 of pregnancy was considered to be the day on which sperm were observed in the smears. The pre-mating exposure condition was continued until pregnancy was confirmed. At this time, the pregnancy exposure 155
regimen began and continued for 6 h a day, 7 days a week through day 20 of gestation. For daffy inhalation exposure, rats were transferred to stainless steel wire mesh racks holding 15 rats in individual 10 × 10 × 20-cm compartments. Two cubic, stainless steel exposure chambers (27 inches/side), with conical upper and lower plenums were used for TCE inhalation exposures. A third cubic chamber and a pentagonal chamber (16 inches/side) were used for filtered air exposures. TCE vapor was generated by heating liquid technical grade solvent containing over 99% TCE* and 0.2% epichlorohydrin. The liquid solvent was contained in a 3-neck round b o t t o m flask heated to 37°C by means of an electric mantle. Compressed air (10 ft. lbs.) was passed through a charcoal filter, swept through the evaporating flask, and introduced into a filtered air stream entering the top plenum of the exposure chamber. Chamber concentrations of TCE were monitored automatically every 13 min throughout the exposure period by a Baseline® gas chromatograph {model 1030A). Exposure concentrations were maintained at the time-weighted average of 1800 + 200 ppm. Previous research has shown this level of exposure to be subnarcotic, and to result in the induction of minor histopathologic lesions in adult male rats with a 4-week exposure period [20]. Pilot studies conducted in this lab indicated t h a t exposure to 1800 ppm of TCE for 3 weeks did n o t significantly alter fertility or mating success. The air flow rate in the chambers was 10.7 feet3/min, and chamber temperature was held to approx. 22°C and never exceeded 27°C.
Maternal and fetal observations All rats were observed daily throughout the exposure period and b o d y weights recorded every 4 days, including days 1 and 21 of gestation. Exposure ended on day 20 of gestation and 15 out of 30 dams/treatment group were sacrificed on day 21 by decapitation. Blood samples were withdrawn by cardiac puncture for SMA-12** determinations in 5 animals/treatment group. Maternal livers were removed, weighed and frozen. Uterine horns were exteriorized through a midline incision in the abdominal wall, and the number of live, dead and resorbed fetuses counted. The umbilical cord of each fetus was clamped and cut distally and the fetus removed. After weighing, fetuses were examined for external anomalies, and their sex was recorded. Four fetuses from each litter were preserved in Bouin's solution for soft tissue analysis by the m e t h o d of Wilson [21]; and 4 fetuses were placed in 95% alcohol for skeletal evaluation by the m e t h o d described by Dawson [22]. Fetuses were selected for analyses so as to represent upper, middle and lower portions of both right and left uterine horns. The livers from the remaining fetuses were removed and frozen in liquid nitrogen for measurement of mixed function oxidase activity. * T h e t r i c h l o r o e t h y l e n e used in this s t u d y was a g e n e r o u s gift of Dow C h e m i c a l C o m p a n y , t r a d e n a m e N E U - T R I ®. E p i c h l o r o h y d r i n is a c o m m o n stabilizer a d d e d to TCE. **Clinical b l o o d test m e a s u r i n g levels o f s e r u m e n z y m e s a n d c o m p o n e n t s indicative of liver a n d k i d n e y d y s f u n c t i o n .
156
Mixed function oxidase enzyme measurements The mixed function oxidase (MFO) enzymes e t h o x y c o u m a r i n dealkylase and ethoxyresorufin dealkylase, indicative of c y t o c h r o m e P-450 and cytochrome P-448 activities, respectively, were measured in maternal and fetal livers, as well as livers of non-pregnant females in each of the treatment groups. A direct fluorimetric procedure reported by Fleischer and Packer [23] was modified to assay enzyme activities in the 9000 g supernatant of adult livers, and 750 g supernatant o f fetal livers, instead of the microsomal pellet. The 7-ethoxyresorufin substrate was obtained from Pierce Chemical Company, R o c k f o r d , IL and the 7-ethoxycoumarin substrate and 7-hydroxycoumarin standard from Aldrich Chemical Company, Milwaukee, WI. Resorufin standard was obtained from Matheson, Coleman and Bell, and the remainder of chemicals used in the assays from Sigma Chemical Company, St. Louis. Pilot assays were performed to optimize concentrations o f substrate, NADPH, and enzyme preparations. The incubation mixture for the ethoxycoumarin dealkylase reaction was prepared b y adding 0.05 ml of a 1 mM ethoxycoumarin solution, 0.05 ml of 9000 g supernatant (or 0.1 ml of the 750 g supernatant) and 1.89 ml of 0.1 M Tris--HC1 (pH 7.6), in a fluorimetric cuvette. After adjusting the excitation and emission wavelengths to 360 and 460 nm, respectively, the percent transmission was recorded prior to adding 0.01 ml of a 10 mM NADPH solution. The reaction was initiated b y adding NADPH and was allowed t o run for 1 min. The incubation mixture for the ethoxyresorufin dealkylase reaction consisted of 0.05 ml of supernatant, and 1.89 ml of 0.1 M Tris--HC1, pH 7.8. After adjusting the excitation and emission wavelengths t o 530 and 585 nm, respectively, the percent transmission was recorded prior to adding 0.01 ml of a 10 mM N A D P H solution. The reaction was allowed to run for 3 min. Postnatal behavioral evaluation The remaining 15 dams/treatment group were allowed to litter. At parturition, all litters were culled to 8 with equal representation o f male and female pups where possible. The dam and litter were housed in polypropylene cages with stainless steel tops until weadlr~g (20 days of age). At weaning, each litter was reduced once again b y random selection of 2 male and 2 female pups and housed in stainless wire b o t t o m cages. Food, water and environmental conditions were as reported above. Activity measurements (10 days o f age) At 10 days of age all offspring were tested for reactivity to transfer from the home cage to a novel environment. The test period was limited t o 10 consecutive 1-min intervals in order to minimize the possibility of thermal stress. Pups were tested in groups of 4 littermates/cage and run simultaneously on 2 electronic Motility Meters (40 FC's). The a m o u n t of general activity was measured b y counting the number of times 40 photocell circuits were interrupted by head movements, pivoting and crawling of the pups. Data were recorded automatically on a printout counter at the end of each 1-min interval. 157
Activity measurements (20 and 1 O0 days of age) One male and female pup/litter were randomly selected and individually tested for a period of 1.5 h in a photocell activity cage measuring 29 × 19 × 13 cm at 20 days of age. Following testing, animals were ear-notched and toeclipped for future identification, and the same animals were then tested on d a y 100. The activity period was divided into 18 consecutive 5-min testing intervals. Ten cages of animals were run simultaneously and monitored by a Kim-1 microprocessor. The pups were assigned to test cages via a balanced design to control for any potential differences in testing conditions across cages. The measure of general activity was again reflected in the number of times photocell circuits were broken by the exploratory behavior exhibited by the rats. Data were recorded automatically by the Kim-1 microprocessor at the end of each 5-min period.
Data analyses The litter was considered to be the experimental unit of analysis, and teratologic data were analyzed by a two-way unweighted means analysis-ofvariance to assess the influence of premating vs. pregnancy exposure [24] with the exception of soft tissue and skeletal data. In order to accomodate the presence of zero values in m a n y of the anomaly categories, KruskalWallis one-way analysis-of-variance by ranks was performed on these latter 2 data sets [25]. Ranks were based on the percentage of the examined litter exhibiting the anomaly. Significant ANOVA effects (P < 0.05) were explored via Newman-Kuels post hoc analysis [26]. Two-way analysis-of-variance with repeated measures was used to assess treatment, sex and trials effects in the behavioral data. Significant effects were explored via Newman-Kuels post hoc analysis. When appropriate, data sets were first transformed via a square-root transformation prior to performing the analysis-of-variance. RESULTS
Teratologic results Table I summarizes maternal effects. Unequal group sizes result from the fact t h a t sperm were n o t always observed in a vaginal smear although mating had occurred. In such cases, if day 1 o f pregnancy could not be accurately determined for the assignment of an animal to a gestational exposure group, the dam was n o t used for teratologic analysis. Mean length o f pregestation exposure was approx. 22 days, and did n o t vary significantly between t r e a t m e n t groups. Female rats exposed to 1800 ppm of TCE before mating and/or during pregnancy failed to exhibit any signs of maternal toxicity. Weight gains throughout pregnancy were normal in relation to filtered air controls. There were no statistically significant differences between treatment conditions or interactions (all F's < 1). Analysis of relative and absolute liver weights indicated no significant treatment effects or interaction of premating and pregnancy exposure (all F's < 1, d f = 1, 39).
158
TABLE I E F F E C T S O F E X P O S U R E TO 1800 p p m T R I C H L O R O E T H Y L E N E ON M A T E R N A L RATS No s i g n i f i c a n t t r e a t m e n t e f f e c t s w e r e o b t a i n e d w i t h t w o - w a y analysis o f variance. Groups a A(++)
B(+--)
Exposure before mating E x p o s u r e during p r e g n a n c y Number of Dams
+ + 12
8
Days P r e ~ x p o s e d B o d y W t (g) Initial Day 1 D a y 21 Liver Wt A b s o l u t e (g) Relative (% o f b o d y w t . )
22 ± 6 b
C(--+)
D(----)
11
12
23 -+ 4
22 -+ 4
22 ± 3
212 ± 39 261 ± 22 362 ± 32
221 ± 39 245 _+ 29 353 -+ 22
221 ± 36 255 ± 26 345 ± 54
204 ± 45 250 ± 23 337 ± 48
12.1 ± 4.6 3.3 ± 1.2
13.2 ± 2.9 3.7 ± .1
13.2 ± 4.9 3.8 ± .9
12.4 -+ 3.5 3.7 ± 1.0
a
F o r i d e n t i f i c a t i o n o f t r e a t m e n t g r o u p s , see M e t h o d s . b M e a n ± S.D.
T A B L E II EVALUATION OF TRICHLOROETHYLENE EMBRYOTOXICITY No s i g n i f i c a n t t r e a t m e n t e f f e c t s w e r e o b t a i n e d w i t h t w o - w a y analysis o f variance. Groupsa A(++) Exposure before mating Exposure during pregnancy N u m b e r o f litters
+ + 12
Corpora lutea/litter I m p l a n t a t i o n sites/litter Live f e t u s e s / l i t t e r F e t a l b o d y w e i g h t (g) R e s o r p t i o n sites/litters a f f e c t e d (%) Litters with 2 or more resorbed S e x ratio, M : F
13.7 13.1 12.2 3.88
± ± ± ±
2.0 b 1.7 2.0 .38
B(+--)
C(-- *)
D(---)
t
--
--
8
11
12
13.1 12.3 11.0 3.97
± ± ± ±
1.4 0.9 0.9 .50
12.3 11.6 10.8 3.87
± ± ± ±
2.6 3.1 3.2 .59
11.9 10.8 10.4 3.90
1.37
1.42
1.33
1.33
3 1:0.9
3 1:0.8
2 1:0.8
1 1:0.9
± 1.7 -+ 2.0 ± 2.1 ± .60
a F o r i d e n t i f i c a t i o n o f t r e a t m e n t groups, see M e t h o d s . b M e a n _+ S.D.
159
T A B L E III S K E L E T A L A N O M A L I E S IN F E T A L R A T S A F T E R M A T E R N A L TRICHLOROETHYLENE EXPOSURE Groups a
Exposure before mating Exposure during pregnancy Number examined I n c o m p l e t e ossification of sternum Missing 2 6 t h a n d 2 7 t h vertebrae S h o r t ribs R u d i m e n t a r y 1 4 t h rib Total
A(++)
B(+ --)
+ + 50(12) b
+
4(2)
---
C(-- +)
D(----)
+
32(8)
43(11)
49(12)
--
8(5)
3(3)
--1(1 )
2(2) 1(1) 2(2)
4(2)
1(1 )
13(8) c
1(1) 1(1 ) 5(4)
a F o r i d e n t i f i c a t i o n o f t r e a t m e n t groups, see M e t h o d s . b N u m b e r of fetuses a f f e c t e d ( n u m b e r of litters affected). CSignificantly e l e v a t e d over filtered air c o n t r o l s , P < 0.05.
Results from blood analysis (SMA-12) performed on maternal rats at sacrifice did not indicate any significant treatment effects on kidney or livel function, and are not shown. Embryotoxicity data are shown in Table II. No significant treatment TABLE IV S O F T T I S S U E A N O M A L I E S IN F E T A L R A T S A F T E R M A T E R N A L TRICHLOROETHYLENE EXPOSURE Groups a A(++)
B(+-)
C(-+)
Exposure before mating Exposure during pregnancy Number examined
+ + 48(12) b
+
F
E n l a r g e d lateral ventricles H e m o r r h a g i c liver Displaced r i g h t o v a r y Enlarged thymus
1( 1 ) I(I )
Total
2(2)
+
32(8)
43(11)
48(12)
8(6)c
3(2)
I(I) --
--
1(1)
aFor identification of treatment groups, see Methods. b N u m b e r of fetuses affected (number of littem affected). CSignificantly elevated over filtered air controls, P < 0.05.
160
D(--)
1(1)
8(6)
4(3)
effects or interactions were found in number of corpora lutea or implantation sites/litter, fetal b o d y weights, resorbed fetuses/litters affected, or sex ratios (all F's < 1, df = 1, 39). Table III summarizes the frequencies of skeletal anomalies in term fetuses. The incidence o f total skeletal anomalies was significantly elevated in group C (P < 0.05), apparently due to the increased incidence o f incomplete ossification of the sternum. When the frequency of this particular anomaly alone was compared in group C vs. group D, no significant difference was found. The incidence of soft tissue anomalies is shown in Table IV. No statistically significant treatment effects were obtained in the analysis of total soft tissue anomalies. The incidence of displaced right ovary, however, was significantly elevated in group C (P < 0.05, one-tailed). The right ovary was displaced approx. 2 mm down from the kidney, while the left ovary was f o u n d dorsocaudal to the kidney in its normal position. Variations in the morphology of other soft tissues, such as enlarged trachea and bronchi, and small bladders, were found distributed across t r e a t m e n t groups, and are n o t shown in Table IV. Variable results were obtained in the MFO enzyme assays which did not correlate with treatment or the state of pregnancy. Consequently, these data will be discussed, b u t n o t shown. TCE stimulated an induction of ethoxycoumarin dealkylase activity (cytochrome P-450) in livers of non-pregnant females in the A and B groups. In contrast, induction was not observed in maternal livers in any o f the t r e a t m e n t groups. The reverse was seen with ethoxyresorufin dealkylase (cytochrome P-448} measurements. Maternal livers from all treatment groups had elevated levels o f activity, while only livers from non-pregnant females in the C group had elevated activity. Measurable levels of ethoxycoumarin dealkylase were present in fetal livers, while ethoxyresorufin dealkylase was undetectable. When values were averaged for the 2 gestationally-exposed groups (A,C) and the gestationally non-exposed groups (B,D), a significant elevation in ethoxycoumarin dealkylase activity was found with gestational exposure to TCE. Postnatal behavioral results Body weight measurements of offspring from birth to 100 days of age are shown in Fig. 1. Two separate analyses were performed on the body weight data because litter weights were taken at birth and 10 days of age, whereas from days 20 to 100 individual weights were recored. The mean weights at birth were 6.4, 6.5, 6.3 and 6.5 for groups A, B, C, and D, respectively. No significant t r e a t m e n t effects were observed in postnatal body weight measurements from birth to 10 days of age. However, from 20 through 100 days of age, TCE exposure in groups A and B (pregestation) was associated with a significant depression in b o d y weights of male and female offspring (P < 0.05 relative to groups C and D). Results from the general activity test at 10 days of age are presented in Fig. 2. Gestationally exposed animals (groups A and C) appeared less active t h a n corresponding controls (groups B and D), but the difference was n o t
161
500 o--oA(+~
400
: : B (+-) a---aC (--+) = : O (--)
.
~
3oo
I,."1-
W
200
IX
IOO
O
~ ,
0
I
I
20
40
i
I
60
=
I
I
80
I00
DAYS OF AGE Fig. 1. Mean body weights of control and TCE-exposed offspring (treatments as indicated in Methods).
statistically significant (P < 0.054). Results from male and female offspring were pooled for presentation purposes due to a lack of sex-related activity levels. Ambulatory response to a noval environment was assessed at 20 and 100 days of age, and data are summarized in Figs. 3 and 4, respectively. No significant treatment effects were obtained in male and female offspring tested under these conditions. In summary, there appears to be a lack of behavioral effects from TCE exposure in offspring from birth to 100 days of age. DISCUSSION The present study was designed to test whether long-term exposure to TCE before mating and during pregnancy caused more adverse reproductive effects than exposure before or during pregnancy alone. In addition, the behavioral effects in postnatal animals following prenatal exposure to TCE was examined for the first time in this study. Significant treatment effects were obtained in regard to elevation of total skeletal anomalies and displaced right ovary in group C with exposure during pregnancy alone. The most frequent skeletal anomaly observed was incomplete ossification of the
162
II
/O-DAY ACTIVITY
9
(/) .J :E
7
,¢ >I--
o - - - o A (++) = --'B (+-) o - - o C (-+) H D (--)
I-(J IX
3
0'
,
0
I
2
,
I
,
I
4 6 TRIALS
,
I
8
,
I
10
Fig. 2. Activity levels of control and TCE-exposed offspring at 10 days of age (treatments as indicated in Methods).
sternum, which is considered to be indicative of delayed skeletal ossification rather than a true malformation. Likewise, the displaced right ovary observed in group C fetuses is most likely a variation-of-normal anomaly rather than a major malformation. It is also possible that this soft tissue anomaly is strain specific and not related to treatment insofar as it has been observed in a number of control as well as treated fetuses in experiments conducted over the past 2 years in this laboratory. No results indicative o f maternal toxicity or embryotoxicity were obtained in any o f the treatment groups. Thus, prenatal exposure to 1800 ppm of trichloroethylene caused an elevation of minor anomalies indicative o f developmental delays in maturation but not of major malformations. Gestational exposure alone led to the most frequent occurrence o f these anomalies rather than long-term exposure before mating and during gestation. Analysis of MFO enzymes in maternal and fetal tissues were conducted to ascertain whether long-term exposure to TCE before mating and during pregnancy resulted in a greater induction o f enzyme activities than short163
A (++)
_
0
I 2
0
I 4
I 6
I 8
(--)
H D
I (0
I (2
.
I (4
I I 16
I , 18
TRIALS
Fig. 3. A c t i v i t y levels of c o n t r o l a n d T C E - e x p o s e d offspring a t 20 d a y s of age ( t r e a t m e n t s as i n d i c a t e d in M e t h o d s ) .
term exposure either before mating or during pregnancy alone. In addition, enzyme activities in pregnant and non-pregnant female livers were compared to determine if metabolism of TCE is altered during pregnancy. Given the reports that TCE is metabolized by phenobarbital-inducible cytochrome P-450 enzymes [27] the elevation of ethoxycoumarin dealkyl9
o..---o A (++) : : B (+-)
a---oC (-+) H D
('--)
5
F I O/
,
0
l
l
2
4
~
l
I
I
I
6
8
I0
12
,
I
I
J
14
16
18
TRIALS
Fig. 4. Activity levels of c o n t r o l a n d T C E - e x p o s e d offspring at t 00 d a y s of age ( t r e a t m e n t s as i n d i c a t e d in Methods).
164
ase activity in TCE-treated non-pregnant livers was expected. The lack of induction in pregnant livers is also consistent with findings of Guenther and Mannering [28] that livers of pregnant rats are resistant to induction of cytochrome P-450 activity, perhaps due to high levels of endogenous steroid hormones which compete with xenobiotics for metabolism. The presence of ethoxyresorufin dealkylase induction in pregnant livers and absence in nonpregnant livers with identical exposures to TCE are difficult t o understand, and are not, to the authors knowledge, interpretable by any similar reports in the literature. The elevation of e t h o x y c o u m a r i n dealkylase activity in fetal livers can be used as an index o f TCE exposure of the fetus, but not necessarily of the potential for fetotoxic effects. In both maternal and fetal livers, exposure before mating and during gestation (Group A) resulted in similar levels of MFO enzymes as exposure during gestation alone (Group C). Thus, longterm exposure to TCE did n o t lead to greater elevation of MFO enzymes than exposure during gestation alone in b o t h maternal and fetal tissues. The conceptus m a y have been exposed to metabolites of TCE, which were n o t measured in this study, for longer periods of time in Group A (++) than Group C ( - +) due to induction of maternal enzymes with premating exposure. However, as the teratologic and neurobehavioral studies indicate, gestational exposure in either the A (++) or C ( - +) group resulted in similar outcomes. Neurobehavioral evaluation of offspring at 10, 20, and 100 days of age indicated a lack of effect on general activity levels. At 10 days of age, gestationally-exposed animals (groups A and C) performed at slightly lower activity levels than animals n o t exposed during gestation. However, this difference was not statistically significant, and the trend was n o t apparent with subsequent testing. Thus, the postnatal behavioral tests revealed no evidence indicative of CNS damage in exposed offspring. A significant depression was observed in postnatal weight gains o f offspring in the premating exposure groups (A and B). Insofar as premating exposure was not found to influence other parameters measured in this study, i.e., maternal toxicity, fetal body weights, skeletal and soft tissue morphology, and activity levels in the offspring, the depression in b o d y weight is difficult to interpret at this time. The offspring from this study are being maintained until 18 m o n t h s of age to assess the p o k n t i a l transplacental carcinogenicity of TCE exposure. The significance of the postnatal weight depression with premating exposure, as well as the skeletal and soft tissue anomalies with gestational exposure, to the overall growth and viability of the offspring will be evaluated in this long-term follow-up. In summary, the results from this study are generally consistent with those reported by Schwetz et al. [18] in t h a t no overt maternal toxicity, e m b r y o t o x i c i t y or teratogenicity were seen. In addition, this study indicates a lack of postnatal behavioral toxicity with exposure of the dam to 1800 opm of TCE. Overall, of the different exoosure periods utilized, exposure during pregnancy alone resulted in the most adverse reproductive effects.
165
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the technical assistance of Ruth Simons, Susanne Tepe and Vince Caruso. REFERENCES 1 E. Browning, Toxicity of Industrial Organic Solvents, H.M.S.O. London, 1953 p. 169. 2 M.R. Truhaut, C. Bondene, N. Phyllich and H. Catella, Arch. Mal. Prof. Med. Tray. Secur. Soc., 28 (1967) 369. 3 NIOSH, Criteria for Recommended Standard-Occupational Exposure to Trichloroethylene, USDHEW, CDC, NIOSH, 1973. 4 M. Kleinfeld and I.R. Tabershaw, Arch. Ind. Hyg. Occup. Med., 10 (1954) 134. 5 E.O. Longley and R. Jones, Arch. Environ. Health, 7 (1963) 249. 6 H.R. Clearfield, Am. J. Dig. Dis., 15 (1970) 851. 7 R.J. Priest and R.C. Horn, Arch. Environ. Health, 11 (1965) 361. 8 C. Albaharry, C. Guyotjeannin, A. Flaisler and P. Thiaucourt, Arch. Mal. Prof., 20 (1959) 421. 9 T.H. Milby, J. Occup. Med., 10 (1968) 252. 10 d.M. Stopps and M. McLaughlin, Am. Ind. Hyg. Assoc. J., 28 (1967) 43. 11 M. Salvini, S. Binaschi and M. Riva, Br. J. Ind. ivied., 28 (1971) 293. 12 C.A. St. Hill, Trans. Soc. Occup. Med., 16 (1966) 6. 13 C.C. Maloof, J. Ind. Hyg. Toxicol., 31 (1949) 295. 14 J. Todd, Br. Med. J., 7 (1954) 439. 15 H.P. Quadland, Ind. Med., 13 (1944) 45. 16 R.S. McBimey, Arch. Ind. Hyg. Occup. IVied., 10 (1954) 130. 17 E. Kunz and R. Isenschmid, Kiln. Monatsbl. Augenheilkd., 94 (1935) 577. 18 B.A. Schwetz, B.K.J. Leong and P.J. Gehring, Toxicol. Appl. Pharmacol., 32 (1975) 84. 19 G. Kimmerle and A. Eben, Arch. Toxikol,, 30 (1973) 127. 20 H. Taylor, J. Ind. Hyg. Toxicol., 18 (1936) 175. 21 J.G. Wilson, Teratology Principles and Techniques, University of Chicago Press, Chicago, 1965, p. 262. 22 A.B. Dawson, Stain Tech., 1 (1926) 123. 23 S. Fleischer and L. Packer, Methods Enzymol., 52 (1977) 372, 24 R.E. Kirk, Experimental Design: Procedures for the Behavioral Sciences, Brooks/Cole Publishing Company, Belmont, California, 1968, p. 120. 25 F.A. Ferguson, Statistical Analysis in Psychology and Education, McGraw-Hili, New York, 1971, p. 182. 26 B.J. Weiner, Statistical Principles in Experimental Design, McGraw-Hill, New York, 1962, p. 286. 27 K.C. Leibman and W.J. McAlister, Jr., J. Pharmacol. Exp. Ther., 157 (1967) 574. 28 T.M. Guenther and G.M. Mannering, Biochem. Pharmacol., 26 (1977) 567.
166
E V A L U A T I O N OF T E R A T O G E N I C I T Y AND B E H A V I O R A L T O X ICIT Y WITH I N H A L A T I O N EXPOSURE O F M A T E R N A L RATS TO TRICHLOROETHYLENE*
MARK A. DORFMUELLER, STEPHEN P. HENNE, RAYMOND G. YORK, ROBERT L. BORNSCHEIN and JEANNE M. MANSON Department of Environmental Health, University of Cincinnati, Cincinnati, OH (U.S.A.) (Received July 25th, 1979) (Accepted October l l t h, 1979)
SUMMARY Female rats were exposed by inhalation to t ri chl oroet hyl ene (TCE) vapors at a c o n c e n t r a t i o n of 1800 -+ 200 p p m t o det erm i ne w h e t h e r exposure before mating and during pregnancy is m o r e detrimental t o reproductive o u t c o m e than exposure either before mating alone or during pregnancy alone. F o u r t r e a t m e n t groups were utilized in a two by t w o factorial design: exposure t o TCE for 2 weeks before mating and during the first 20 days o f pregnancy; TCE before mating and filtered air during pregnancy; filtered air before mating and TCE during pregnancy; and filtered air before and during pregnancy. Significant elevations in skeletal and soft tissue anomalies, indicative o f developmental delay in m at ur a t i on rather than teratogenesis, were observed in the group exposed during pregnancy alone. The mixed funct i on oxidase enzymes, e t h o x y c o u m a r i n and e t h o x y r e s o r u f i n , indicative o f cytoch r o me P-450 and P-448 activities, respectively, were measured in maternal and fetal livers, as well as livers of non-pregnant females, and showed variable levels o f activity n o t u n i f o r m l y related t o t r e a t m e n t or pregnancy. Behavioral evaluation o f offspring indicated a lack of t r e a t m e n t effect in tests o f general activity levels at 10, 20 and 100 days o f age. However, a reduct i on in postnatal b o d y weights was seen in offspring from m ot hers with pregestational exposure. No results indicative o f treatment-related maternal toxicity, e m b r y o t o x i c i t y , severe teratogenicity or significant behavioral deficits were obtained in any o f the t r e a t m e n t groups. *Supported by NIEHS grants No. ES 01601 and No. 3-P30-ES00159 Center for the Study of the Human Environment. Address Correspondence to: Dr. Jeanne M. Manson, Department of Environmental Health, University of Cincinnati, 3223 Eden Avenue, Cincinnati, OH 45267, U.S.A. Abbreviations: MFO, mixed function oxidase; TCE, trichloroethylene.
153
INTRODUCTION Trichloroethylene (TCE) is an unsaturated chlorinated hydrocarbon (molecular formula CI:C=CHC1) widely used in industrial and commercial products. Over 9(Wo of the TCE produced in the United States is utilized as a liquid or vapor cleaning solvent in industrial, automotive, aircraft and aerospace degreasing operations. The remaining percentage is used as a component in many common consumer products including cleansers for automobiles, buffing solutions, spot removers, rug cleaners, pesticides and paints [1,2]. The current Federal occupational exposure limit to TCE is 100 p p m determined as a time-weighted average for an 8-h exposure period with a ceiling limit of 200 p p m [3]. There has been no exact determination of the amount of TCE that is fatal to humans. However, there are numerous case reports of workers w h o have died as a result of TCE exposure. In the best documented cases [4,5] exposUre levels were estimated to be between 1700 and 3300 ppm. Prior to death, the workmen complained of nausea, drowsiness, dizziness, and vomiting. Several investigators [6,7] have reported hepatic necrosis and nephropathy resulting from inhalation of TCE contained in aerosol products. Chronic exposure to TCE has been reported t o result in elevated levels of serum transaminases indicative of damage to the liver parenchyma [8]. However, Milby [9] f o u n d no evidence of abnormal liver or kidney function (SGOT, SGPT, erythrocyte sedimentation rate) in a paint stripping machine operator exposed to 280 ppm TCE. Several studies have reported behavioral alterations in workers exposed to TCE. Stopps and McLaughlin [10] found a progressive decline in psychom o t o r performance with increasing TCE exposure over a range of 100--500 ppm. Similarly, Salvini et al. [11] noted a decrease in performance on a manual dexterity test for subjects exposed to 110 ppm of TCE for t w o 4-h sessions. They also found significant decreases in performance on a perception test, the Wechsler m e m o r y scale and a complex reaction time test. Other reported behavioral changes following acute exposure to TCE include diplopia [12,13], paranoid psychosis and hallucinations [14]. Chronic exposure to TCE has resulted in CNS disturbances [15], loss of tactile sense [16] and in one case, total blindness [17]. TCE was one o f several solvents tested for teratogenic potential by Schwetz et al. [18]. Rats and mice were exposed b y inhalation for 7 h/day on days 6--15 of gestation to 300 p p m of TCE. Dams were sacrificed at term, and evaluation of material toxicity, e m b r y o t o x i c i t y and teratogenicity yielded negative results. The short-term exposure period utilized in this study constitutes the conventional test of teratogenicity which m a y n o t be adequate to predict the teratogenic potential of TCE. Kimmerle and Eben [19] demonstrated that the pharmacokinetic behavior of TCE after acute exposure differs from that after subacute exposure. These investigators found that repeated exposure resulted in continuous elevation of the toxic metabolite, trichloroethanol, in the blood while levels of the parent c o m p o u n d 154
remained relatively stable. Short~term exposure from days 6--15 of gestation could lead to an underestimation of the teratogenicity of TCE insofar as metabolic activation systems generating toxic metabolites may not be induced during the organogenesis period when the conceptus is most susceptible to teratogenic insult. Thus, one aim of the present study was to assess the reproductive consequences of exposure to TCE both before mating and during pregnancy compared to before mating alone or during pregnancy alone. The currently accepted protocol for teratology testing (i.e., sacrifice of pregnant dams at term following exposure during the organogenesis period, and examination o f maternal and fetal tissue) is appropriate for investigation o f structural anomalies in term fetuses, but not of the functional integrity of the offspring. In the latter case, postnatal behavioral evaluation m a y be a more sensitive index of overall functional capacity. As noted previously several studies have found behavioral alterations as a result of chronic or acute exposure to TCE in adult humans. However, systematic investigation o f the behavioral effects in postnatal animals following prenatal exposure to TCE have y e t to be conducted. Thus, a second aim o f the present study was to assess the effects of maternal exposure to TCE prior to and/or during pregnancy on the behavioral development and function of the offspring. METHODS Animals and exposure conditions
One hundred and t w e n t y virgin female Long-Evans hooded rats were purchased from Charles River Breeding Laboratories. Animals were ear-notched for individual identification and toe-clipped for treatment group identification, and quarantined for 2 weeks after arrival. They were singly housed in stainless steel wire b o t t o m cages in a temperature (23°C) and light-controlled r o o m (12:12 light--clark cycle, light commencing at 7 a.m.). Standard laboratory rat chow (Purina 5001) and distilled water were available ad lib. except during residence in exposure chambers. At the beginning of the experiment, female rats weighing approx. 210 g were randomly assigned to 1 of 4 treatment groups, with 30 rats per group as follows: A (++) - TCE exposure before mating and during pregnancy; B ( + - ) -- TCE exposure before mating, filtered air during pregnancy; C ( - +) -- Filtered air before mating, TCE during pregnancy; D ( - - ) -- Filtered air before mating and during pregnancy; The pre-mating exposure was conducted for 6 h a day, 5 days a week for 2 weeks. Following the last day of exposure, the mating regimen began. Two estrous females were placed with 1 breeder male of the same strain overnight.Vaginal smears were conducted the following morning prior to exposure. Day 1 of pregnancy was considered to be the day on which sperm were observed in the smears. The pre-mating exposure condition was continued until pregnancy was confirmed. At this time, the pregnancy exposure 155
regimen began and continued for 6 h a day, 7 days a week through day 20 of gestation. For daffy inhalation exposure, rats were transferred to stainless steel wire mesh racks holding 15 rats in individual 10 × 10 × 20-cm compartments. Two cubic, stainless steel exposure chambers (27 inches/side), with conical upper and lower plenums were used for TCE inhalation exposures. A third cubic chamber and a pentagonal chamber (16 inches/side) were used for filtered air exposures. TCE vapor was generated by heating liquid technical grade solvent containing over 99% TCE* and 0.2% epichlorohydrin. The liquid solvent was contained in a 3-neck round b o t t o m flask heated to 37°C by means of an electric mantle. Compressed air (10 ft. lbs.) was passed through a charcoal filter, swept through the evaporating flask, and introduced into a filtered air stream entering the top plenum of the exposure chamber. Chamber concentrations of TCE were monitored automatically every 13 min throughout the exposure period by a Baseline® gas chromatograph {model 1030A). Exposure concentrations were maintained at the time-weighted average of 1800 + 200 ppm. Previous research has shown this level of exposure to be subnarcotic, and to result in the induction of minor histopathologic lesions in adult male rats with a 4-week exposure period [20]. Pilot studies conducted in this lab indicated t h a t exposure to 1800 ppm of TCE for 3 weeks did n o t significantly alter fertility or mating success. The air flow rate in the chambers was 10.7 feet3/min, and chamber temperature was held to approx. 22°C and never exceeded 27°C.
Maternal and fetal observations All rats were observed daily throughout the exposure period and b o d y weights recorded every 4 days, including days 1 and 21 of gestation. Exposure ended on day 20 of gestation and 15 out of 30 dams/treatment group were sacrificed on day 21 by decapitation. Blood samples were withdrawn by cardiac puncture for SMA-12** determinations in 5 animals/treatment group. Maternal livers were removed, weighed and frozen. Uterine horns were exteriorized through a midline incision in the abdominal wall, and the number of live, dead and resorbed fetuses counted. The umbilical cord of each fetus was clamped and cut distally and the fetus removed. After weighing, fetuses were examined for external anomalies, and their sex was recorded. Four fetuses from each litter were preserved in Bouin's solution for soft tissue analysis by the m e t h o d of Wilson [21]; and 4 fetuses were placed in 95% alcohol for skeletal evaluation by the m e t h o d described by Dawson [22]. Fetuses were selected for analyses so as to represent upper, middle and lower portions of both right and left uterine horns. The livers from the remaining fetuses were removed and frozen in liquid nitrogen for measurement of mixed function oxidase activity. * T h e t r i c h l o r o e t h y l e n e used in this s t u d y was a g e n e r o u s gift of Dow C h e m i c a l C o m p a n y , t r a d e n a m e N E U - T R I ®. E p i c h l o r o h y d r i n is a c o m m o n stabilizer a d d e d to TCE. **Clinical b l o o d test m e a s u r i n g levels o f s e r u m e n z y m e s a n d c o m p o n e n t s indicative of liver a n d k i d n e y d y s f u n c t i o n .
156
Mixed function oxidase enzyme measurements The mixed function oxidase (MFO) enzymes e t h o x y c o u m a r i n dealkylase and ethoxyresorufin dealkylase, indicative of c y t o c h r o m e P-450 and cytochrome P-448 activities, respectively, were measured in maternal and fetal livers, as well as livers of non-pregnant females in each of the treatment groups. A direct fluorimetric procedure reported by Fleischer and Packer [23] was modified to assay enzyme activities in the 9000 g supernatant of adult livers, and 750 g supernatant o f fetal livers, instead of the microsomal pellet. The 7-ethoxyresorufin substrate was obtained from Pierce Chemical Company, R o c k f o r d , IL and the 7-ethoxycoumarin substrate and 7-hydroxycoumarin standard from Aldrich Chemical Company, Milwaukee, WI. Resorufin standard was obtained from Matheson, Coleman and Bell, and the remainder of chemicals used in the assays from Sigma Chemical Company, St. Louis. Pilot assays were performed to optimize concentrations o f substrate, NADPH, and enzyme preparations. The incubation mixture for the ethoxycoumarin dealkylase reaction was prepared b y adding 0.05 ml of a 1 mM ethoxycoumarin solution, 0.05 ml of 9000 g supernatant (or 0.1 ml of the 750 g supernatant) and 1.89 ml of 0.1 M Tris--HC1 (pH 7.6), in a fluorimetric cuvette. After adjusting the excitation and emission wavelengths to 360 and 460 nm, respectively, the percent transmission was recorded prior to adding 0.01 ml of a 10 mM NADPH solution. The reaction was initiated b y adding NADPH and was allowed t o run for 1 min. The incubation mixture for the ethoxyresorufin dealkylase reaction consisted of 0.05 ml of supernatant, and 1.89 ml of 0.1 M Tris--HC1, pH 7.8. After adjusting the excitation and emission wavelengths t o 530 and 585 nm, respectively, the percent transmission was recorded prior to adding 0.01 ml of a 10 mM N A D P H solution. The reaction was allowed to run for 3 min. Postnatal behavioral evaluation The remaining 15 dams/treatment group were allowed to litter. At parturition, all litters were culled to 8 with equal representation o f male and female pups where possible. The dam and litter were housed in polypropylene cages with stainless steel tops until weadlr~g (20 days of age). At weaning, each litter was reduced once again b y random selection of 2 male and 2 female pups and housed in stainless wire b o t t o m cages. Food, water and environmental conditions were as reported above. Activity measurements (10 days o f age) At 10 days of age all offspring were tested for reactivity to transfer from the home cage to a novel environment. The test period was limited t o 10 consecutive 1-min intervals in order to minimize the possibility of thermal stress. Pups were tested in groups of 4 littermates/cage and run simultaneously on 2 electronic Motility Meters (40 FC's). The a m o u n t of general activity was measured b y counting the number of times 40 photocell circuits were interrupted by head movements, pivoting and crawling of the pups. Data were recorded automatically on a printout counter at the end of each 1-min interval. 157
Activity measurements (20 and 1 O0 days of age) One male and female pup/litter were randomly selected and individually tested for a period of 1.5 h in a photocell activity cage measuring 29 × 19 × 13 cm at 20 days of age. Following testing, animals were ear-notched and toeclipped for future identification, and the same animals were then tested on d a y 100. The activity period was divided into 18 consecutive 5-min testing intervals. Ten cages of animals were run simultaneously and monitored by a Kim-1 microprocessor. The pups were assigned to test cages via a balanced design to control for any potential differences in testing conditions across cages. The measure of general activity was again reflected in the number of times photocell circuits were broken by the exploratory behavior exhibited by the rats. Data were recorded automatically by the Kim-1 microprocessor at the end of each 5-min period.
Data analyses The litter was considered to be the experimental unit of analysis, and teratologic data were analyzed by a two-way unweighted means analysis-ofvariance to assess the influence of premating vs. pregnancy exposure [24] with the exception of soft tissue and skeletal data. In order to accomodate the presence of zero values in m a n y of the anomaly categories, KruskalWallis one-way analysis-of-variance by ranks was performed on these latter 2 data sets [25]. Ranks were based on the percentage of the examined litter exhibiting the anomaly. Significant ANOVA effects (P < 0.05) were explored via Newman-Kuels post hoc analysis [26]. Two-way analysis-of-variance with repeated measures was used to assess treatment, sex and trials effects in the behavioral data. Significant effects were explored via Newman-Kuels post hoc analysis. When appropriate, data sets were first transformed via a square-root transformation prior to performing the analysis-of-variance. RESULTS
Teratologic results Table I summarizes maternal effects. Unequal group sizes result from the fact t h a t sperm were n o t always observed in a vaginal smear although mating had occurred. In such cases, if day 1 o f pregnancy could not be accurately determined for the assignment of an animal to a gestational exposure group, the dam was n o t used for teratologic analysis. Mean length o f pregestation exposure was approx. 22 days, and did n o t vary significantly between t r e a t m e n t groups. Female rats exposed to 1800 ppm of TCE before mating and/or during pregnancy failed to exhibit any signs of maternal toxicity. Weight gains throughout pregnancy were normal in relation to filtered air controls. There were no statistically significant differences between treatment conditions or interactions (all F's < 1). Analysis of relative and absolute liver weights indicated no significant treatment effects or interaction of premating and pregnancy exposure (all F's < 1, d f = 1, 39).
158
TABLE I E F F E C T S O F E X P O S U R E TO 1800 p p m T R I C H L O R O E T H Y L E N E ON M A T E R N A L RATS No s i g n i f i c a n t t r e a t m e n t e f f e c t s w e r e o b t a i n e d w i t h t w o - w a y analysis o f variance. Groups a A(++)
B(+--)
Exposure before mating E x p o s u r e during p r e g n a n c y Number of Dams
+ + 12
8
Days P r e ~ x p o s e d B o d y W t (g) Initial Day 1 D a y 21 Liver Wt A b s o l u t e (g) Relative (% o f b o d y w t . )
22 ± 6 b
C(--+)
D(----)
11
12
23 -+ 4
22 -+ 4
22 ± 3
212 ± 39 261 ± 22 362 ± 32
221 ± 39 245 _+ 29 353 -+ 22
221 ± 36 255 ± 26 345 ± 54
204 ± 45 250 ± 23 337 ± 48
12.1 ± 4.6 3.3 ± 1.2
13.2 ± 2.9 3.7 ± .1
13.2 ± 4.9 3.8 ± .9
12.4 -+ 3.5 3.7 ± 1.0
a
F o r i d e n t i f i c a t i o n o f t r e a t m e n t g r o u p s , see M e t h o d s . b M e a n ± S.D.
T A B L E II EVALUATION OF TRICHLOROETHYLENE EMBRYOTOXICITY No s i g n i f i c a n t t r e a t m e n t e f f e c t s w e r e o b t a i n e d w i t h t w o - w a y analysis o f variance. Groupsa A(++) Exposure before mating Exposure during pregnancy N u m b e r o f litters
+ + 12
Corpora lutea/litter I m p l a n t a t i o n sites/litter Live f e t u s e s / l i t t e r F e t a l b o d y w e i g h t (g) R e s o r p t i o n sites/litters a f f e c t e d (%) Litters with 2 or more resorbed S e x ratio, M : F
13.7 13.1 12.2 3.88
± ± ± ±
2.0 b 1.7 2.0 .38
B(+--)
C(-- *)
D(---)
t
--
--
8
11
12
13.1 12.3 11.0 3.97
± ± ± ±
1.4 0.9 0.9 .50
12.3 11.6 10.8 3.87
± ± ± ±
2.6 3.1 3.2 .59
11.9 10.8 10.4 3.90
1.37
1.42
1.33
1.33
3 1:0.9
3 1:0.8
2 1:0.8
1 1:0.9
± 1.7 -+ 2.0 ± 2.1 ± .60
a F o r i d e n t i f i c a t i o n o f t r e a t m e n t groups, see M e t h o d s . b M e a n _+ S.D.
159
T A B L E III S K E L E T A L A N O M A L I E S IN F E T A L R A T S A F T E R M A T E R N A L TRICHLOROETHYLENE EXPOSURE Groups a
Exposure before mating Exposure during pregnancy Number examined I n c o m p l e t e ossification of sternum Missing 2 6 t h a n d 2 7 t h vertebrae S h o r t ribs R u d i m e n t a r y 1 4 t h rib Total
A(++)
B(+ --)
+ + 50(12) b
+
4(2)
---
C(-- +)
D(----)
+
32(8)
43(11)
49(12)
--
8(5)
3(3)
--1(1 )
2(2) 1(1) 2(2)
4(2)
1(1 )
13(8) c
1(1) 1(1 ) 5(4)
a F o r i d e n t i f i c a t i o n o f t r e a t m e n t groups, see M e t h o d s . b N u m b e r of fetuses a f f e c t e d ( n u m b e r of litters affected). CSignificantly e l e v a t e d over filtered air c o n t r o l s , P < 0.05.
Results from blood analysis (SMA-12) performed on maternal rats at sacrifice did not indicate any significant treatment effects on kidney or livel function, and are not shown. Embryotoxicity data are shown in Table II. No significant treatment TABLE IV S O F T T I S S U E A N O M A L I E S IN F E T A L R A T S A F T E R M A T E R N A L TRICHLOROETHYLENE EXPOSURE Groups a A(++)
B(+-)
C(-+)
Exposure before mating Exposure during pregnancy Number examined
+ + 48(12) b
+
F
E n l a r g e d lateral ventricles H e m o r r h a g i c liver Displaced r i g h t o v a r y Enlarged thymus
1( 1 ) I(I )
Total
2(2)
+
32(8)
43(11)
48(12)
8(6)c
3(2)
I(I) --
--
1(1)
aFor identification of treatment groups, see Methods. b N u m b e r of fetuses affected (number of littem affected). CSignificantly elevated over filtered air controls, P < 0.05.
160
D(--)
1(1)
8(6)
4(3)
effects or interactions were found in number of corpora lutea or implantation sites/litter, fetal b o d y weights, resorbed fetuses/litters affected, or sex ratios (all F's < 1, df = 1, 39). Table III summarizes the frequencies of skeletal anomalies in term fetuses. The incidence o f total skeletal anomalies was significantly elevated in group C (P < 0.05), apparently due to the increased incidence o f incomplete ossification of the sternum. When the frequency of this particular anomaly alone was compared in group C vs. group D, no significant difference was found. The incidence of soft tissue anomalies is shown in Table IV. No statistically significant treatment effects were obtained in the analysis of total soft tissue anomalies. The incidence of displaced right ovary, however, was significantly elevated in group C (P < 0.05, one-tailed). The right ovary was displaced approx. 2 mm down from the kidney, while the left ovary was f o u n d dorsocaudal to the kidney in its normal position. Variations in the morphology of other soft tissues, such as enlarged trachea and bronchi, and small bladders, were found distributed across t r e a t m e n t groups, and are n o t shown in Table IV. Variable results were obtained in the MFO enzyme assays which did not correlate with treatment or the state of pregnancy. Consequently, these data will be discussed, b u t n o t shown. TCE stimulated an induction of ethoxycoumarin dealkylase activity (cytochrome P-450) in livers of non-pregnant females in the A and B groups. In contrast, induction was not observed in maternal livers in any o f the t r e a t m e n t groups. The reverse was seen with ethoxyresorufin dealkylase (cytochrome P-448} measurements. Maternal livers from all treatment groups had elevated levels o f activity, while only livers from non-pregnant females in the C group had elevated activity. Measurable levels of ethoxycoumarin dealkylase were present in fetal livers, while ethoxyresorufin dealkylase was undetectable. When values were averaged for the 2 gestationally-exposed groups (A,C) and the gestationally non-exposed groups (B,D), a significant elevation in ethoxycoumarin dealkylase activity was found with gestational exposure to TCE. Postnatal behavioral results Body weight measurements of offspring from birth to 100 days of age are shown in Fig. 1. Two separate analyses were performed on the body weight data because litter weights were taken at birth and 10 days of age, whereas from days 20 to 100 individual weights were recored. The mean weights at birth were 6.4, 6.5, 6.3 and 6.5 for groups A, B, C, and D, respectively. No significant t r e a t m e n t effects were observed in postnatal body weight measurements from birth to 10 days of age. However, from 20 through 100 days of age, TCE exposure in groups A and B (pregestation) was associated with a significant depression in b o d y weights of male and female offspring (P < 0.05 relative to groups C and D). Results from the general activity test at 10 days of age are presented in Fig. 2. Gestationally exposed animals (groups A and C) appeared less active t h a n corresponding controls (groups B and D), but the difference was n o t
161
500 o--oA(+~
400
: : B (+-) a---aC (--+) = : O (--)
.
~
3oo
I,."1-
W
200
IX
IOO
O
~ ,
0
I
I
20
40
i
I
60
=
I
I
80
I00
DAYS OF AGE Fig. 1. Mean body weights of control and TCE-exposed offspring (treatments as indicated in Methods).
statistically significant (P < 0.054). Results from male and female offspring were pooled for presentation purposes due to a lack of sex-related activity levels. Ambulatory response to a noval environment was assessed at 20 and 100 days of age, and data are summarized in Figs. 3 and 4, respectively. No significant treatment effects were obtained in male and female offspring tested under these conditions. In summary, there appears to be a lack of behavioral effects from TCE exposure in offspring from birth to 100 days of age. DISCUSSION The present study was designed to test whether long-term exposure to TCE before mating and during pregnancy caused more adverse reproductive effects than exposure before or during pregnancy alone. In addition, the behavioral effects in postnatal animals following prenatal exposure to TCE was examined for the first time in this study. Significant treatment effects were obtained in regard to elevation of total skeletal anomalies and displaced right ovary in group C with exposure during pregnancy alone. The most frequent skeletal anomaly observed was incomplete ossification of the
162
II
/O-DAY ACTIVITY
9
(/) .J :E
7
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o - - - o A (++) = --'B (+-) o - - o C (-+) H D (--)
I-(J IX
3
0'
,
0
I
2
,
I
,
I
4 6 TRIALS
,
I
8
,
I
10
Fig. 2. Activity levels of control and TCE-exposed offspring at 10 days of age (treatments as indicated in Methods).
sternum, which is considered to be indicative of delayed skeletal ossification rather than a true malformation. Likewise, the displaced right ovary observed in group C fetuses is most likely a variation-of-normal anomaly rather than a major malformation. It is also possible that this soft tissue anomaly is strain specific and not related to treatment insofar as it has been observed in a number of control as well as treated fetuses in experiments conducted over the past 2 years in this laboratory. No results indicative o f maternal toxicity or embryotoxicity were obtained in any o f the treatment groups. Thus, prenatal exposure to 1800 ppm of trichloroethylene caused an elevation of minor anomalies indicative o f developmental delays in maturation but not of major malformations. Gestational exposure alone led to the most frequent occurrence o f these anomalies rather than long-term exposure before mating and during gestation. Analysis of MFO enzymes in maternal and fetal tissues were conducted to ascertain whether long-term exposure to TCE before mating and during pregnancy resulted in a greater induction o f enzyme activities than short163
A (++)
_
0
I 2
0
I 4
I 6
I 8
(--)
H D
I (0
I (2
.
I (4
I I 16
I , 18
TRIALS
Fig. 3. A c t i v i t y levels of c o n t r o l a n d T C E - e x p o s e d offspring a t 20 d a y s of age ( t r e a t m e n t s as i n d i c a t e d in M e t h o d s ) .
term exposure either before mating or during pregnancy alone. In addition, enzyme activities in pregnant and non-pregnant female livers were compared to determine if metabolism of TCE is altered during pregnancy. Given the reports that TCE is metabolized by phenobarbital-inducible cytochrome P-450 enzymes [27] the elevation of ethoxycoumarin dealkyl9
o..---o A (++) : : B (+-)
a---oC (-+) H D
('--)
5
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,
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l
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6
8
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12
,
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Fig. 4. Activity levels of c o n t r o l a n d T C E - e x p o s e d offspring at t 00 d a y s of age ( t r e a t m e n t s as i n d i c a t e d in Methods).
164
ase activity in TCE-treated non-pregnant livers was expected. The lack of induction in pregnant livers is also consistent with findings of Guenther and Mannering [28] that livers of pregnant rats are resistant to induction of cytochrome P-450 activity, perhaps due to high levels of endogenous steroid hormones which compete with xenobiotics for metabolism. The presence of ethoxyresorufin dealkylase induction in pregnant livers and absence in nonpregnant livers with identical exposures to TCE are difficult t o understand, and are not, to the authors knowledge, interpretable by any similar reports in the literature. The elevation of e t h o x y c o u m a r i n dealkylase activity in fetal livers can be used as an index o f TCE exposure of the fetus, but not necessarily of the potential for fetotoxic effects. In both maternal and fetal livers, exposure before mating and during gestation (Group A) resulted in similar levels of MFO enzymes as exposure during gestation alone (Group C). Thus, longterm exposure to TCE did n o t lead to greater elevation of MFO enzymes than exposure during gestation alone in b o t h maternal and fetal tissues. The conceptus m a y have been exposed to metabolites of TCE, which were n o t measured in this study, for longer periods of time in Group A (++) than Group C ( - +) due to induction of maternal enzymes with premating exposure. However, as the teratologic and neurobehavioral studies indicate, gestational exposure in either the A (++) or C ( - +) group resulted in similar outcomes. Neurobehavioral evaluation of offspring at 10, 20, and 100 days of age indicated a lack of effect on general activity levels. At 10 days of age, gestationally-exposed animals (groups A and C) performed at slightly lower activity levels than animals n o t exposed during gestation. However, this difference was not statistically significant, and the trend was n o t apparent with subsequent testing. Thus, the postnatal behavioral tests revealed no evidence indicative of CNS damage in exposed offspring. A significant depression was observed in postnatal weight gains o f offspring in the premating exposure groups (A and B). Insofar as premating exposure was not found to influence other parameters measured in this study, i.e., maternal toxicity, fetal body weights, skeletal and soft tissue morphology, and activity levels in the offspring, the depression in b o d y weight is difficult to interpret at this time. The offspring from this study are being maintained until 18 m o n t h s of age to assess the p o k n t i a l transplacental carcinogenicity of TCE exposure. The significance of the postnatal weight depression with premating exposure, as well as the skeletal and soft tissue anomalies with gestational exposure, to the overall growth and viability of the offspring will be evaluated in this long-term follow-up. In summary, the results from this study are generally consistent with those reported by Schwetz et al. [18] in t h a t no overt maternal toxicity, e m b r y o t o x i c i t y or teratogenicity were seen. In addition, this study indicates a lack of postnatal behavioral toxicity with exposure of the dam to 1800 opm of TCE. Overall, of the different exoosure periods utilized, exposure during pregnancy alone resulted in the most adverse reproductive effects.
165
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the technical assistance of Ruth Simons, Susanne Tepe and Vince Caruso. REFERENCES 1 E. Browning, Toxicity of Industrial Organic Solvents, H.M.S.O. London, 1953 p. 169. 2 M.R. Truhaut, C. Bondene, N. Phyllich and H. Catella, Arch. Mal. Prof. Med. Tray. Secur. Soc., 28 (1967) 369. 3 NIOSH, Criteria for Recommended Standard-Occupational Exposure to Trichloroethylene, USDHEW, CDC, NIOSH, 1973. 4 M. Kleinfeld and I.R. Tabershaw, Arch. Ind. Hyg. Occup. Med., 10 (1954) 134. 5 E.O. Longley and R. Jones, Arch. Environ. Health, 7 (1963) 249. 6 H.R. Clearfield, Am. J. Dig. Dis., 15 (1970) 851. 7 R.J. Priest and R.C. Horn, Arch. Environ. Health, 11 (1965) 361. 8 C. Albaharry, C. Guyotjeannin, A. Flaisler and P. Thiaucourt, Arch. Mal. Prof., 20 (1959) 421. 9 T.H. Milby, J. Occup. Med., 10 (1968) 252. 10 d.M. Stopps and M. McLaughlin, Am. Ind. Hyg. Assoc. J., 28 (1967) 43. 11 M. Salvini, S. Binaschi and M. Riva, Br. J. Ind. ivied., 28 (1971) 293. 12 C.A. St. Hill, Trans. Soc. Occup. Med., 16 (1966) 6. 13 C.C. Maloof, J. Ind. Hyg. Toxicol., 31 (1949) 295. 14 J. Todd, Br. Med. J., 7 (1954) 439. 15 H.P. Quadland, Ind. Med., 13 (1944) 45. 16 R.S. McBimey, Arch. Ind. Hyg. Occup. IVied., 10 (1954) 130. 17 E. Kunz and R. Isenschmid, Kiln. Monatsbl. Augenheilkd., 94 (1935) 577. 18 B.A. Schwetz, B.K.J. Leong and P.J. Gehring, Toxicol. Appl. Pharmacol., 32 (1975) 84. 19 G. Kimmerle and A. Eben, Arch. Toxikol,, 30 (1973) 127. 20 H. Taylor, J. Ind. Hyg. Toxicol., 18 (1936) 175. 21 J.G. Wilson, Teratology Principles and Techniques, University of Chicago Press, Chicago, 1965, p. 262. 22 A.B. Dawson, Stain Tech., 1 (1926) 123. 23 S. Fleischer and L. Packer, Methods Enzymol., 52 (1977) 372, 24 R.E. Kirk, Experimental Design: Procedures for the Behavioral Sciences, Brooks/Cole Publishing Company, Belmont, California, 1968, p. 120. 25 F.A. Ferguson, Statistical Analysis in Psychology and Education, McGraw-Hili, New York, 1971, p. 182. 26 B.J. Weiner, Statistical Principles in Experimental Design, McGraw-Hill, New York, 1962, p. 286. 27 K.C. Leibman and W.J. McAlister, Jr., J. Pharmacol. Exp. Ther., 157 (1967) 574. 28 T.M. Guenther and G.M. Mannering, Biochem. Pharmacol., 26 (1977) 567.
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