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Paternal exposure of rabbits to lead: Behavioral deficits in offspring
Neurotoxicology
and Teratology, Vol. 19, No. 3, pp. 191-198,1997 Copyright 0 1997 Elsevier Science Inc.
Printed in the USA. All rights reserved 0892-0362/97 $17.00 + .oO ELSEVIER
PIISO892-0362(96)00221-S
Paternal Exposure of Rabbits to Lead: Behavioral Deficits in Offspring B. K. NELSON,*
W. J. MOORMAN, S. M. SCHRADER, AND EDWARD F. KRIEG, JR.
PETER
B. SHAW
Division of Biomedical and Behavioral Science, National Institute for Occupational Safety and Health, Cincinnati, OH 45226 Received
1 August
1996; Accepted
26 November
1996
NELSON, B. K., W. J. MOORMAN, S. M. SCHRADER, P. B. SHAW AND E. F. KRIEG, JR. Paternal exposure ofrabbits ro lead: Behavioral deficits in offwrirw. NEUROTOXICOL TERATOL 19(3) 191-198,1997.-Paternal exoosures to exogenous agents have been;eported?dprovduce a variety of developmental defectsin the offspring. In experimental animals, these effects include decreased litter size and weight, increased stillbirth and neonatal death, birth defects, tumors, and functional/behavioral abnormalities-some of these effects being transmitted to the second and third generations. The majority of experimental studies assessing nervous system function of offspring following paternal exposures have utilized rats as the experimental animal, but other species can be used. The National Toxicology Program (NTP) has initiated studies to validate the rabbit as an animal model for human reproductive toxicity, because rabbits are the smallest laboratory animal from which ejaculates can be collected repeatedly. An important part of reproductive toxicology is assessment of the reproductive ability of males following exposure, as well as developmental and functional assessment of their offspring. This article describes a pilot study and a main study to investigate the feasibility of using rabbits to assess the functional effects of paternal exposure to lead. The pilot study included seven male rabbits per group exposed for 15 weeks to lead acetate sufficient to produce 0,50, or 110 pg/dl blood lead. The main study included 15 male rabbits per group exposed for 15 weeks to lead acetate to produce 0,20,40, and 80 pg/dl blood lead. At the conclusion of the exposure, male rabbits were mated with unexposed females. These females carried their litters to term, delivered, and reared their own offspring. The offspring were weighed at 5, 10, 15, 20, 25, 30, and some at 35 days of age. They were also tested for exploratory activity in a standard figure-eight “maze” for 30 min/day on days 15, 20, 25, and 30. A second assessment of exploratory behavior, along with a simple test of aversive conditioning, was attempted in the pilot study, but was judged not to be suitable for the main study. Of the 21 male rabbits that were mated in the pilot study, 16 produced viable litters (6/7, 6/7, and 4/7 in control, low- and high-lead groups, respectively), with a mean number of 6 live births/litter in each treatment group (range 2-8). Of the 60 rabbits mated in the main study, 57 produced litters, and two rabbits died giving birth. Significant postnatal deaths were observed in all groups, with about one half of the offspring dying before testing was initiated at day 15. There were no treatment-related effects on offspring weight gain through weaning. The data suggest that paternal lead exposure of rabbits may reduce figure-eight activity on day 25, the time of peak activity in the offspring. 0 1997 Elsevier Science Inc. Figure-8 activity Lead Male-mediated effects
Paternal
exposure
Reproductive
AS is well established, developmental toxicity (manifested by embryo/fetal death, malformations, growth impairment, and functional deficits) can be produced by exposure of the maternal organism to exogenous agents during development. About 3% of all children born in the United States have a major malformation detectable at birth; some 67% of infants have developmental disorders detectable by 1 year of age; and 12-14% of children have developmental disorders detectable by school
Requests for reprints should be addressed to B. K. Nelson, Fax: (513) 533-8596; E-mail: [email protected]
NIOSH
toxicology
Neurobehavioral
Rabbit
age (33). A separate survey suggests that about 17% of children have had one or more developmental disabilities up through age 17 (6). About 70% of these developmental disorders are of unknown etiology. Historically, embryos prior to implantation were thought to be relatively immune to the production of developmental disorders (4,66). Evidence suggested that exposure of this early conceptus to an adverse influence either killed the con-
C-24,4676
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Parkway,
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OH 45226. Tel: (513) 533-8178;
192 ceptus or the embryo withstood this influence with no detectable damage. It is now apparent, however, that exposure during preimplantation periods to certain agents can produce developmental defects in the offspring [see reviews (29.33. 52,55)]. Various agents can produce preimplantation effects. including opiates (14,15). nickel chloride (57) medroxyprogesterone acetate (13) methylnitrosurea (28,59), ethylene oxide (20) nonionizing (radiofrequency) radiation (34), and ionizing radiation (44,52). Extending the implications of gestational exposure to subsequent generations is a recent report that ethylnitosourea administered during embryogenesis to female mice produced mutations in the primordial germ cells of the male offspring (64). These mutations manifested as reduced fertility when these males were mated with untreated females, and increased malformations in the next generation. The mechanism(s) by which most developmental toxicants function are unknown. It is apparent, however, that genetic mechanisms may be operational, particularly for those agents that act prior to organogenesis. Consequently, it is not surprising that paternal exposures may also induce developmental defects in the offspring. Developmental defects reported to result from paternal exposures include decreased litter size and weight, increased stillbirth and neonatal death, birth defects, tumors, and behavioral/neurochemical abnormalitiessome of these effects being transmitted to the second and third generations (2,3,8,10,12,14-18,3&32,37,42,43,47,48,60,61). Several possible mechanisms by which exposure of the male may result in disorders in the offspring have been postulated, including genetic (germ cell) alterations, toxic or epigenetic effects, seminal fluid transfer of toxicants, and hormonal alterations in the males, potentially leading to infertility and germ cell loss (8,67). Lead is one of the most studied of any developmental toxicant [e.g., (5,11,46)]. Lead has also been investigated for effects on the male, as well as male-mediated effects on the offspring, in several studies [e.g., (5,19,36,45,58)]. For example, male and female rats were intubated with 500 mg/kg lead acetate from 30-90 days of age (7). At 90 days of age, these males and females were mated together or to control males and females; lead-exposed females continued exposure throughout gestation and lactation. Offspring from these matings (17 pups/group) were tested for learning in a water T-maze beginning at 30 days of age. The three lead-exposed groups (viz., maternal only, paternal only, or maternal plus paternal) made more errors in the maze than the controls, but did not differ from one another. However, offspring in the maternal plus paternal exposure group had longer swimming times than those in either maternally or paternally exposed groups, and those in the latter groups had longer swimming times than the controls. Thus, either maternal or paternal exposure exerted deleterious effects on the offspring, but maternal plus paternal exposure had the most severe effects. In another study, 90-day-old Sprague-Dawley rats were exposed for 70 days to 0.3% lead acetate in drinking water (blood lead of 58 pgidl ), or to 5 mg/cms; lead oxide via inhalation (aerosol) for 6 h/day, 5 days per week (blood lead of 51 kg/d]) (45). In addition to the standard reproductive endpoint assessments, these investigators mated the exposed males to unexposed females. Some of the mated females were sacrificed on gestation day 20 and examined for corpora lutea of pregnancy, implantation sites. resorptions, and number and sex ratio of fetuses (none of these parameters was affected by lead exposure). Other pregnant females were allowed to deliver and rear their litters, and the offspring were followed until they were 90 days old. At that time. some of the male off-
NELSON
ET AL.
spring were examined for reproductive parameters and the others were mated to determine fertility. The growth pattern of the exposed males did not differ from that of controls, and the only reproductive parameter affected was a reduction in the number of spermatozoa in the cauda epididymides. In spite of that reduction, there was no effect on the fertility of the offspring. Others have investigated male-mediated developmental disorders in rats (19). Paternal lead-induced changes have been observed in early gene expression in early embryos, as well as alterations in hippocampal development of late fetal and early neonatal rats. Taken together, these studies suggest the need for additional research on paternally mediated effects of lead-particularly because lead is an ubiquitous pollutant.
FEASIBILITY OF USING RABBITS TO DETECT MALE-MEDIATED EFFECTS
As described above, several chemicals have demonstrated paternally mediated developmental toxicity in experimental animals. The majority of experimental studies assessing function of offspring following maternal or paternal exposures have utilized rats as the experimental animal (21), but other species are used. For example, the National Toxicology Program (NTP) has initiated studies to validate the rabbit as an animal model for assessing male reproductive toxicity. The rabbit was chosen as the animal model because it is the smallest laboratory animal from which an ejaculate can be routinely collected. Ejaculated semen, assessing accessory sex gland secretions, more closely simulates human studies than does collection of epididymal sperm from rodent models. Being able to collect ejaculates allows for multiple samples. utilizing a longitudinal study design with experimental animals. An important part of reproductive toxicology is assessing the reproductive ability of males following exposure, as well as functional assessment of their offspring. One of the NTP validation studies is investigating the reproductive toxicology of lead administered to rabbits (3840,53,54,62,68). The merits of using rabbits in neurobehavioral testing have been described (25). A major point is that rabbits may provide an excellent model for behavioral teratology because maternal rabbits provide little care for their young-hence there is less concern about the need for fostering. Rabbits build a nest from available materials, including their own fur. However, once the babies are born, the doe leaves the pups and returns to nurse only once every 24 h. When she returns to nurse, the doe positions herself over the nest, not giving the pups any assistance in suckling. She does not brood them, clean them, or retrieve them to the nest. After lo-15 min of nursing, the doe departs until the next day. The neonates are born without fur, with eyes and outer ears sealed, and with poor motor coordination. Their eyes open around days 10-12. They develop thermoregulatory ability about day 15, around the time they start to leave the nest. By the time of weaning at around 5 weeks of age, their weight has increased from approximately 40 g at birth to 400-500 g. Given this information about young rabbits. the projected use of rabbits in male reproductive toxicology, and the availability of lead-exposed paternal rabbits, we conducted two exploratory studies. This article describes our attempt to use lead-exposed rabbits in a preliminary feasibility study to determine the utility of rabbit offspring for behavioral testing of offspring after paternal exposure. Based on the results of this preliminary study, the second phase consisted of the main
PATERNAL
EXPOSURE
OF RABBITS
study utilizing a range of doses numbers of exposed rabbits.
of lead,
193
TO LEAD along
with higher
METHOD
Details of the experimental design, dosing procedures, collection and analysis of blood and semen samples, histopathology of testes, and hormone analyses are presented separately for the preliminary studies (38,39,53,68) and the main studies (40,54,62). Briefly, in a project approved by our IACUC, sexually mature male and female Dutch Belted rabbits were purchased (6-7 months old; Hazelton Research Products; Aberdeen, MD). They were maintained in an AAALAC-approved facility in temperature-controlled rooms with tap water and Purina high-fiber rabbit chow (125 g/day) and a 12/12 light/ dark cycle. Lead acetate (O-3.85 mg/kg on Monday, Wednesday, and Friday) was administered SC to groups of rabbits (following 5 weeks of preexposure baseline acclimationitesting) for 15 weeks (through six cycles of the seminiferous epithelium to ensure that all sperm were formed under plateau lead exposure conditions). Weekly blood lead levels were measured by atomic absorption spectroscopy with Zeeman background corrections in a Centers for Disease Control and Prevention certified laboratory (a national reference laboratory). After the first 5 weeks of lead administration, the blood levels had reached a plateau, and were stable. In the preliminary phase, seven rabbits/group were given lead to reach target blood levels at Control (i 5), 50, and 110 kg/dl. In the main study, groups of 15 male rabbits/group were administered lead acetate sufficient to produce target blood levels at Control (< 5). 20,40, and 80 kgidl. In the preliminary study, males in the Control (actual mean = 0.2; limit of detection = l.l), 50 (actual mean = 5.5.9), and 110 (actual mean = 125.6) pg/dl blood lead levels were mated with virgin female rabbits within a week after the last lead injection. Because of the nature of the other testing involved with these males. all matings (3 groups X 7 rabbits per group = 21 rabbits) were accomplished in 2 days. The does were housed in 25 X 20 X 16-in. stainless steel cages and not disturbed, other than weekly weighing and cage cleaning, until expected parturition when litter boxes and nest material were provided. In the main study, males in the Control (actual mean = 0.6), 20 (actual mean = 24.6), 40 (actual mean = 38.9), and 80 (actual mean = 71.2) kg/d1 blood lead levels were mated with virgin female rabbits within a week after the last lead injection. To achieve minimal spread in birth of the young rabbits, yet still maintain the other desired testing, all 60 males (4 groups X 15 males) were mated over a period of 10 days. At the time of expected parturition, rabbits were checked each morning and evening for delivery. On the day of birth (day 0), the live offspring were counted. Otherwise, to minimize handling (and resulting probability of the mother killing the neonates), there was minimal contact with the pups (daily examination for number alive/dead, weekly cage changes). They were weighed individually on postnatal days 5,10, 15,20, 25,30, and (some on) 35. Testing was performed by personnel blind to the treatment groups of the rabbits being tested. Standard figure-eight rodent activity monitors (stainless steel, wire mesh flooring; Omnitech Electronics, Inc.; Columbus, OH) were used to assess exploratory motor activity (1). Pilot studies suggested that motor activity was minimal before the eyes were open (day 10-12). and peaked at around 25 days of age. By about 35 days of age, the rabbits’ size may have adversely affected their activity because of the size of the figure-eight corridors. Consequently, rabbits were tested on
days 15. 20, 25, and 30. Testing was for a 30-min test period, with data (number of entries into area of a new photocell, and total activity counts) recorded every 5 min. Another test that purportedly assesses exploratory activity involves the consumption of novel liquids [e.g., (22,35,65)], and this test was attempted in the preliminary study. This test also allowed us a simple test of learning (conditioned taste aversion). Patterned after the testing paradigm of Weinberg et al. (65), rabbits were tested on days 20-31. Days 20-25 served as the acquisition phase for the animals learning to consume the novel substance. On day 25, an aversive stimulus was administered to the rabbits following consumption of the novel liquid. Days 26-31 served as the recovery phase. From days 20-25, a 30% (w/w) sucrose solution was provided ad lib in bottles for 1 h at about 1200 h (acquisition phase; following the figure-eight testing on days 20 and 25). For this hour, littermates were placed together in a 22 X 12.5 X g-in. hanging polypropylene cage, and all fluid loss from the bottle was presumed to result from consumption by the young rabbits. After this test on day 25, each rabbit was injected IP with 7.5 ml/kg lithium chloride (LiCl; 0.4 M; Sigma Chemical Co.; St. Louis, MO), which serves as an aversive stimulus in animals (aversive conditioning phase). From days 26-31, rabbits were given the same liquid at approximately the same hours as from days 20-25 (recovery phase). (Because of the variability in response and lack of significant results, this test was not used in the main study.) The data were analyzed using multivariate analysis of variance (MANOVA) for repeated measures. In the preliminary study, even though a control and two dose levels of lead were included, loss of litters in the high-lead group rendered that group inappropriate to include in the statistical analyses (see the Results section). The null hypothesis (H,) in this test is that the means for all of the days under consideration, for a given dependent variable, are the same for all comparison groups. Rejection of H, implies that the means are different for at least 1 day. We used an F-test calculated from Wilk’s lambda as the criterion in our tests. We examined four dependent variables in the preliminary study. These included mean litter weight on days 5, 10, 15,20, 25, 30, and 35; mean litter “activity” and “entries” in the fig-
Offspring Weights - Preliminary Study 700 Legend
Jr
FIG. 1. Weight gain in offspring of rabbits paternally exposed
to lead (control, 50, and 100 kg/dl). Data from all three groups are presented, although excessive deaths in the high-lead group prohibited its inclusion in the statislical analysis (preliminary study).
194
NELSON
ET AL.
Sucrose Solution Consumed
Figure-8 Activity Chang.
by Agm (Acdvlty)
FIG. 3. Sucrose consumption in offspring exposed to lead (control, 50 t.qg/dl) (preliminary
of rabbits study).
paternally
300 Legend mo-
*~l.Y***.
n*b I..&
-----
Lowl...d
-
conbo(
low-lead, and control groups, respectively), with a mean number of six live births in each treatment group (range 2-8). Gestation length was not affected (30 -C 1 day). Significant postnatal deaths were observed in all groups, but were highest in the high-lead group. By 10 days of age, only two litters had surviving pups in the high lead group (two litters had no surviving pups, one litter had one pup, and one litter had six pups, for a total of seven pups); six litters had an average of 3.2 pups/litter in the low lead group (two litters had two pups,
M)-
0.8
summary activity data in offspring of rabbits paternally exposed to lead (control, 50, and 100 pg/dl). Data from all three groups arc presented, although excessive deaths in the high-lead
FIG.
2. Figure-eight
c~~~~~_~~_~~~~~~~~_~~~~~~~_
I-
group prohibited its inclusion in the statistical analysis. Top = number of entries. Bottom = total activity counts (preliminary study).
,’ V B
litter sucrose consumption for pairs of days 20 and 21,22 and 23,24 and 25, 26 and 27, 28 and 29, and 30 and 31 (this pairing was necessary to afford sufficient degrees of freedom to carry out the test). In the main study, ANOVA was used to test for day, period, and lead effects, and their interactions. The litter was the unit of analysis, with mean values for each litter calculated prior to analysis. MANOVA was used to test within-litter effects (day, period, and their interactions). An F-value calculated from Wilk’s lambda was used as the test statistic. Between-litter effects and contrasts were done with F-tests. Contrasts were done to compare lead values if there was a significant main effect or interaction involving lead. These calculations were done with SAS7 Version 6.11 (SAS Institute, Inc., Cary, NC) we-eight
monitors
on days
15,20,
25, and 30; and mean
RESULTS
_’
,
0.6
0.5
k? 1
0.4
a 0.3
0.2
0.1
0.0 0
5
10
15
26
25
80
Day of Age
Preliminary Study Of the 21 male rabbits that were mated in the preliminary study, 16 produced viable litters (4/7, 6/7, 6/7 in high-lead,
FIG. 4. Cumulative mean proportions dead of offspring of rabbits paternally exposed to lead (control, 20,40, and 80 kg/dl) as a function of age (main study).
PATERNAL
EXPOSURE
OF RABBITS
195
TO LEAD
DayofAge
-IS --20 ---
25
..-.-
30
400
22 E
*B 300 3
4
a
c
200
I
loo
5
x)
15
20
25
30
1
2
3
4
5
6
Period
FIG. 5. Weight gain (g) in offspring of rabbits paternally exposed lead (control,
20,40,
and 80 pgidl)
to
(main study).
DayofAge \
litter had three pups, one litter had five pups, one litter had seven pups, for a total of 19 pups); and six litters had an average of three pups/litter in the control group (one litter had two pups, one litter had three pups, two litters had four pups, and one litter had five pups, for a total of 18 pups). As discussed above, the high-lead group had only one litter with more than one pup per litter. Consequently, we did not include this group in the statistical analyses, but the data are included in the first two figures. Figure 1 presents the offspring weight gain through 3.5 days of age in the preliminary study, but no significant treatment-related effects were observed. Figure 2 presents the preliminary study figure-eight activity data, where there was evidence of possible differences between groups (Fig. 2A = number of entries [lambda = 0.21; F(4, 5) = 4.7, p = 0.061, Fig. 2B = total activity counts [lambda = 0.61; F(4,S) = 6.4, p = 0.031). Figure 3 presents the consumption of sucrose solution from days 2&31, where there was no evidence of group differences. one
‘\
\
\
\
\
-IS --20 --- 25 .----.---30
\ ‘1
Main Study
Of the 60 does mated in this study, 57 delivered litters (15/15, 15115, 13/15, and 14/15 in the control to high-lead group. respectively). Thus, the lead levels used in this study did not affect the reproductive ability of the male rabbits (mean implants per litter = 6.6. 7.6, 7.2, and 7.6 pups per litter, for the control to high-lead group, respectively). Gestation length was not affected (30 5 1 day). Two of the does died giving birth (one in the control group and one in the lowlead group). As in the preliminary study, nearly one half of the offspring were either dead at birth, or died within about 1 week after birth. Figure 4 shows the mean proportion of pups dead on days 0 to 30. The main effect of day was significant
1
2
4
3
5
6
Period FIG. 6. Overall mean period areas (top) and activity counts offspring (combined treatment and 30) (main study).
(5 min) by test age entries into new (bottom) of the figure-eight in rabbit groups) by day of age (day 15,20,25,
[lambda = 0.25; F(14,38) = 8.2, p = O.OOOl]. The main effect of lead approached significance (p = 0.07). Figure 5 shows the mean body weight of the pups as a function of day of age. The main effect of age was significant
NELSON
196 30
25 .-fi? : 20 w z ?I n 15 E ii! s
1
ET AL.
35
Lead (pg/dL)
I
CIO iQz 20 m 40 BZZ% 80
30 i y) 25 F 2 o 20 1 0 .z
10
r”
8
15-
5 =
IO-
5
0 15
Day
20
25
FIG. 7. Mean figure-eight “entries” (left) and “activity” (right) in offspring of rabbits paternally to lead (control. 20.40. and 80 kgidl) hy day ot’age (day 15.20.25. and 30) (main study).
[lambda = 0.03; F(S. 32) = 208.2. p = O.OOOl], but there were no treatment-related effects. Figure 6 shows the significant day X period interactions for entries [lambda = 0.21: F(lS, 21) = 5.2,~ = 0.0003] and activity [lambda = 0.33; F(15, 21) = 2.8. p = O.Ol]. As illustrated, young rabbits were most active during the first periods tested in the figure-eight at all ages. However. activity during the first half of the test was lowest on day 15, and highest on day 25. Figure 7 shows the significant lead X day interactions for entries [lambda = 0.59; F(9. X0) = 2.2, p = 0.031 and activity [lambda = 0.54: F(9. 80) = 2.6, 11 = O.Ol]. For entries: the mean (? SEM) of the 80 *g/d1 lead group (17.2 i 2.2) was significantly less than the mean of the 0 group (23.3 + 1.8) on day 25 (j = 0.03); the mean of the 40 kg/d1 lead group (16.2 5 1.7) was significantly less than the mean of the 0 group on day 25 (~9 = 0.01); and the mean of the 40 kg/d1 lead group (17.3 ? 1.7) was significantly greater than the mean of the 20 pgidl lead group (11.9 -t 1.7) on day 30 (JI = 0.03). For activity: the mean of the 80 kg/d1 lead group (23.5 ? 2.8) was nearly significantly less than the mean of the 0 group (30.6 5 2.3) on day 25 (p = 0.05): the mean of the 40 pgidl lead group (21.4 5 2.2) was significantly less than the mean of the 0 group on day 25 (p = 0.005): and the mean of the 40 kg/d1 lead group was significantly less than the mean of the 20 p.g/dl lead group (28.8 ? 2.2) on day 25 b = 0.02). DISCIJSSION
The data in the highest lead group (110 pgidl blood lead. preliminary study) are suggestive of an adverse effect of lead on fertility (417 litters). However, no effect on fertility was noted at 75-80 pg/dl blood lead, and additional research will be needed to verify the antifertility effects at the highest level. Additional research will also be necessary to determine if the incidence of postnatal deaths in rabbits in the high-lead group (110 pgidl blood lead. preliminary study) was treatment related, as the sample sizes were too small to make definitive statements. The next lower dose (75-80 kg/d1 blood lead) did not appear to produce effects on postnatal deaths. (Although the incidence of postnatal deaths seems high, con-
30
35
Day exposed
versations with laboratory rabbit breeders indicate that this rate is not unusual for prima gravida females.) Our data suggest that the levels of lead we used do not affect offspring weight gain, at least through the time of weaning. This lack of effects on fertility or on weight gain is consistent with the lack of effects seen in male rats after paternal lead exposure (45). The pattern of activity seen in the young rabbits (viz. an increase in activity to a peak and then a decrease in activity with age) is typical of that seen in rats, with the peak somewhat dependent on the activity measuring device [e.g., (l)]. It appears that the peak we observed was a real peak observed in behavior (the consistency seen in three “replications,” viz., a few pilot litters, those in the preliminary study. as well as those in the main study, is quite striking), although it is possible that the size of the rabbits may have affected their activity at the oldest age tested. The pattern of high activity during the first period of test, decreasing to little or no activity by the end of testing (Fig. 6). is typical of that reported in figure-eight testing [e.g., (9)]. In fact, the lJS EPA guideline on motor activity assessments specifies that the duration of testing allow motor activity to approach asymptotic levels by the last 20% of the session (63). The hypoactivity we observed after paternal lead exposure (day 25: reflected both in the number of entries into new areas as well as in the overall activity: Fig. 7) is similar to that reported after maternal lead exposure [e.g., (46)], although hyperactivity and no effects on activity have also been reported (1 I). As the latter investigators point out, different measures of activity do not necessarily correlate with one another. Furthermore. activity levels can be influenced by species, age, sex, estrous cycle, time of day, novelty of environment. experience. and food deprivation. etc. We did not find literature references to consumption of sucrose or the use of lithium chloride in rabbits. The 30% sucrose solution and dose of lithium chloride we selected were based upon references using rats. If rabbits are to be used extensively for this kind of research, more detailed investigations should assess the appropriateness of the sucrose and lithium chloride levels we used. Extensive research has been conducted with neonatal rabbits. particularly investigating the development of olfactory
PATERNAL
EXPOSURE
OF RABBITS
TO LEAD
197
function and odor conditioning [e.g., (23-27,56)]. Because of the more extensive testing that would be involved, and the clustering of births in our study, we did not investigate olfactory function. However, utilization of the data and experience of Hudson and Distel would add additional testing capabilities to those we used. A rabbit model for studying the prenatal effects of cocaine has recently been described (41) along with conditioning and discrimination learning (49-51). In summary, these data suggest the need for more research into paternally mediated effects, in experimental animals as well as in humans. Based on the present study, we believe that rabbits can be used to detect postnatal functional deficits following paternal exposures to exogenous agents. However, the high rate of postnatal deaths in rabbits, as opposed to that seen in rats, makes the use of rabbits less favorable for routine screening than rats. Nonetheless, when rabbits would make
better models for detecting the adverse reproductive effects of the specific chemical or physical agents being tested, rabbits can be used for the postnatal studies as well. Our data suggest no effects of paternal exposure below 75-80 kg/d1 blood lead on fertility, survivability of offspring, or weight gain through weaning in rabbits. It does appear, however, that exploratory activity may be affected in the offspring at and above 40 kg/d1 blood lead.
ACKNOWLEDGEMENTS We are grateful for the support of Dr. Bob Chapin and NIEHSI NTP throughout these studies (Interagency Agreement #YOl-ES40266 “Validation of the Rabbit Model for Assessing Reproductive Toxicants”). We thank the animal caretaker staff, particularly Alissa Christman. for their excellent care of the young rabbits in the studies.
REFERENCES in behavioral teratology. In: Riley, E. P.; 1. Adams. J.: Methods Vorhees. C. V.. eds. Handbook of behavioral teratology. New York: Plenum Press: 19X6:67-97. 2. Adams, P. M.; Shabrawy, 0.: Legator, M. S.: Male-transmitted developmental and neurobehavioral deficits. Teratogenesis Carcinog. Mutagen. 4:149-169: 1984. 3. Auroux, M. R.: Dulioust, E. J.: Nawar. N. N.; Yacoub, S. G.; Mayaux. M. J.: Schwartz, D.; David. G.: Antimitotic drugs in the male rat: Behavioral abnormalities in the second generation. J. Androl. 9:153-159; 1988. 4. Austin. C. R.: Embryo transfer and sensitivity to teratogenesis. Nature 244~333-334; 1973. update: Lead. Teratology 50:367-373: 5. Bellinger, D.: Teratogen 1994. M.: Prevalence and 6. Boyle, C. A.; Decoufle, P.; Yeargig-Allsopp, health impact of developmental disabilities in US children. Pediatrics 93:399-403; 1994. 7. Brady, K.; Herrera, Y.: Zenick, H.: Influence of parental lead exposure on subsequent learning ability of offspring. Pharmacol. Biochem. Behav. 3:561-565; 1975. Reprod. Toxicol. 7:38. Colie. C. F.: Male mediated teratogenesis. 9; 1993. 9. Crofton, K. M.; Howard. J. L.; Moser, V. C.: Gill, M. W.: Reiter, L. W.: Tilson, H. A.: MacPhail, R. C.: Interlaboratory comparison of motor activity experiments: Implications for neurotoxicological assessments. Neurotoxicol. Teratol. 13:599-609: 1991. 10. Davis, D. L.; Friedler, G.; Mattison, D.; Morris. R.: Male-mediated teratogenesis and other reproductive effects: Biologic and epidemiologic findings and a plea for clinical research. Reprod. Toxicol. 6:289-292; 1992. 11. Davis, J. M.: Otto, D. A.: Weil, D. E.; Grant, L. D.: The comparative developmental neurotoxicity of lead in humans and animals. Neurotoxicol. Teratol. 12:215-229: 1990. E. J. B.; Nawar, N. Y.: Yacoub. S. G.; Ebel, A. B.; 12. Dulioust. Kcmpf. E. H.; Auroux. M. R.: Cyclophosphamide in the male rat: New pattern of anomalies in the third generation. J. Androl. 10:296-303; 1989. H.; Hagele, M.: Teratogenic effects of 13. Eibs. H-G.: Speilmann. cyproterone acetate and medroxyprogesterone treatment during the pre and postimplantation period of mouse embryos. I. Teratology 2527-36; 1982. effects of opiates. In: Dancis, J.; Hwang. 14. Friedler, G.: Long-term J. C., eds. Pcrinatal pharmacology: Problems and priorities. New York: Raven Press: 1974:207-216. 15. Friedler. G.: Pregestational administration of morphine sulfate to female mice: Long-term effects on the development of subsequent progeny. J. Pharmacol. Exp. Ther. 205:33-39: 1978. exposure to xenobiotic 16. Fricdler, G.: Effects of limited paternal agents on the development of progeny. Neurobehav. Toxicol. Teratol. 7:739-743: 1985.
toxicology: Male-mediated effects. 17 Friedler, G.: Developmental In: Paul, M., ed. Occupational and environmental reproductive hazards: A guide to clinicians. Baltimore. MD: Williams & Wilkins; 1992:52-59. H. S.: Behavioral effects in offspring of 18. Friedler, G.; Wheeling, male mice injected with opiates prior to mating. Protracted effects of perinatal drug dependence. Pharmacol. Biochem. Behav. 1 l(Suppl.):23-28: 1979. R. E.; Silbergeld, E. K.: Male-mediated reproductive 19 Gandley. toxicity: Effects on the nervous system of offspring. In: Mattison, D. R.: Olshan, A. F., eds. Male-mediated developmental toxicity. New York: Plenum Press; 1994:141-151. W. M.; Rutledge, J. C.: Cain, K. T.: Hughes, L. A.; 20 Generoso, Braden, P. W.: Exposure of female mice to ethylene oxide within hours after mating leads to fetal malformations and death. Mutat. Res. 176~2699274; 1987. of the 21 Goldey, E. S.; Tilson, H. A.; Crofton. K. M.: Implications use of neonatal birth weight, growth, viability, and survivability data for predicting developmental neurotoxicity: A survey of the literature. Neurotoxicol. Teratol. 17:313-332: 1995. M. B.; Smotherman, W. P.; Levine, S.: Early olfactory 22 Hennessy, enrichment enhances later consumption of novel substances. Physiol. Behav. 19:481-483; 1977. R.; Distel, H.: Nipple location by newborn rabbits: 23 Hudson. Behavioral evidence for pheromonal guidance. Behaviour 85: 260-275; 1983. R.; Distel, H.: Nipple-search pheromone in rabbits: 24 Hudson. Dependence on season and reproductive status. J. Comp. Physiol. [A] 155:13-17; 1984. 25 Hudson, R.; Distel. H.: The potential of the newborn rabbit for behavioral teratological research. Neurobehav. Toxicol. Teratol. X:209-21 2: 1986. 26 Hudson, R.: Distel, H.: Temporal pattern of suckling in rabbit pups: A model of circadian synchrony between mother and young. In: Reppert, S. M., ed. Research in perinatal medicine vol. IX: Development of circadian rhythmicity and photoperiodism in mammals. London: Perinatology Press: 19X9:83-102. 27 Hudson, R.; Distel, H.: Development of olfactory function in newborn rabbits: Inborn and learned responses. Oslo: ISOT X, Proc. 10th Int. Symp. Olfaction Taste. GCS A/S. 1990:216-225. P. M.: Long-term effects of exposure to methylnitro28 Iannaccone, surea on blastocysts following transfer to surrogate female mice. Cancer Res. 44:2785-2789; 1984. 29 Iannaccone, P. M.; Bossert, N. L.; Connelly, C. S.: Disruption of embryonic and fetal development due to preimplantation chemical insults: A critical review. Am. J. Obstet. Gynecol. 157:4764X4; 1987. 30 Joffe, J. M.: Influence of drug exposure of the father on perinatal outcome. Clin. Perinatol. 6:2ll36; 1979. M.: Milkovic. K.: Progeny of male rats 31 Joffe. J. M.: Peruzovic,
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and Teratology, Vol. 19, No. 3, pp. 191-198,1997 Copyright 0 1997 Elsevier Science Inc.
Printed in the USA. All rights reserved 0892-0362/97 $17.00 + .oO ELSEVIER
PIISO892-0362(96)00221-S
Paternal Exposure of Rabbits to Lead: Behavioral Deficits in Offspring B. K. NELSON,*
W. J. MOORMAN, S. M. SCHRADER, AND EDWARD F. KRIEG, JR.
PETER
B. SHAW
Division of Biomedical and Behavioral Science, National Institute for Occupational Safety and Health, Cincinnati, OH 45226 Received
1 August
1996; Accepted
26 November
1996
NELSON, B. K., W. J. MOORMAN, S. M. SCHRADER, P. B. SHAW AND E. F. KRIEG, JR. Paternal exposure ofrabbits ro lead: Behavioral deficits in offwrirw. NEUROTOXICOL TERATOL 19(3) 191-198,1997.-Paternal exoosures to exogenous agents have been;eported?dprovduce a variety of developmental defectsin the offspring. In experimental animals, these effects include decreased litter size and weight, increased stillbirth and neonatal death, birth defects, tumors, and functional/behavioral abnormalities-some of these effects being transmitted to the second and third generations. The majority of experimental studies assessing nervous system function of offspring following paternal exposures have utilized rats as the experimental animal, but other species can be used. The National Toxicology Program (NTP) has initiated studies to validate the rabbit as an animal model for human reproductive toxicity, because rabbits are the smallest laboratory animal from which ejaculates can be collected repeatedly. An important part of reproductive toxicology is assessment of the reproductive ability of males following exposure, as well as developmental and functional assessment of their offspring. This article describes a pilot study and a main study to investigate the feasibility of using rabbits to assess the functional effects of paternal exposure to lead. The pilot study included seven male rabbits per group exposed for 15 weeks to lead acetate sufficient to produce 0,50, or 110 pg/dl blood lead. The main study included 15 male rabbits per group exposed for 15 weeks to lead acetate to produce 0,20,40, and 80 pg/dl blood lead. At the conclusion of the exposure, male rabbits were mated with unexposed females. These females carried their litters to term, delivered, and reared their own offspring. The offspring were weighed at 5, 10, 15, 20, 25, 30, and some at 35 days of age. They were also tested for exploratory activity in a standard figure-eight “maze” for 30 min/day on days 15, 20, 25, and 30. A second assessment of exploratory behavior, along with a simple test of aversive conditioning, was attempted in the pilot study, but was judged not to be suitable for the main study. Of the 21 male rabbits that were mated in the pilot study, 16 produced viable litters (6/7, 6/7, and 4/7 in control, low- and high-lead groups, respectively), with a mean number of 6 live births/litter in each treatment group (range 2-8). Of the 60 rabbits mated in the main study, 57 produced litters, and two rabbits died giving birth. Significant postnatal deaths were observed in all groups, with about one half of the offspring dying before testing was initiated at day 15. There were no treatment-related effects on offspring weight gain through weaning. The data suggest that paternal lead exposure of rabbits may reduce figure-eight activity on day 25, the time of peak activity in the offspring. 0 1997 Elsevier Science Inc. Figure-8 activity Lead Male-mediated effects
Paternal
exposure
Reproductive
AS is well established, developmental toxicity (manifested by embryo/fetal death, malformations, growth impairment, and functional deficits) can be produced by exposure of the maternal organism to exogenous agents during development. About 3% of all children born in the United States have a major malformation detectable at birth; some 67% of infants have developmental disorders detectable by 1 year of age; and 12-14% of children have developmental disorders detectable by school
Requests for reprints should be addressed to B. K. Nelson, Fax: (513) 533-8596; E-mail: [email protected]
NIOSH
toxicology
Neurobehavioral
Rabbit
age (33). A separate survey suggests that about 17% of children have had one or more developmental disabilities up through age 17 (6). About 70% of these developmental disorders are of unknown etiology. Historically, embryos prior to implantation were thought to be relatively immune to the production of developmental disorders (4,66). Evidence suggested that exposure of this early conceptus to an adverse influence either killed the con-
C-24,4676
191
Columbia
Parkway,
Cincinnati,
OH 45226. Tel: (513) 533-8178;
192 ceptus or the embryo withstood this influence with no detectable damage. It is now apparent, however, that exposure during preimplantation periods to certain agents can produce developmental defects in the offspring [see reviews (29.33. 52,55)]. Various agents can produce preimplantation effects. including opiates (14,15). nickel chloride (57) medroxyprogesterone acetate (13) methylnitrosurea (28,59), ethylene oxide (20) nonionizing (radiofrequency) radiation (34), and ionizing radiation (44,52). Extending the implications of gestational exposure to subsequent generations is a recent report that ethylnitosourea administered during embryogenesis to female mice produced mutations in the primordial germ cells of the male offspring (64). These mutations manifested as reduced fertility when these males were mated with untreated females, and increased malformations in the next generation. The mechanism(s) by which most developmental toxicants function are unknown. It is apparent, however, that genetic mechanisms may be operational, particularly for those agents that act prior to organogenesis. Consequently, it is not surprising that paternal exposures may also induce developmental defects in the offspring. Developmental defects reported to result from paternal exposures include decreased litter size and weight, increased stillbirth and neonatal death, birth defects, tumors, and behavioral/neurochemical abnormalitiessome of these effects being transmitted to the second and third generations (2,3,8,10,12,14-18,3&32,37,42,43,47,48,60,61). Several possible mechanisms by which exposure of the male may result in disorders in the offspring have been postulated, including genetic (germ cell) alterations, toxic or epigenetic effects, seminal fluid transfer of toxicants, and hormonal alterations in the males, potentially leading to infertility and germ cell loss (8,67). Lead is one of the most studied of any developmental toxicant [e.g., (5,11,46)]. Lead has also been investigated for effects on the male, as well as male-mediated effects on the offspring, in several studies [e.g., (5,19,36,45,58)]. For example, male and female rats were intubated with 500 mg/kg lead acetate from 30-90 days of age (7). At 90 days of age, these males and females were mated together or to control males and females; lead-exposed females continued exposure throughout gestation and lactation. Offspring from these matings (17 pups/group) were tested for learning in a water T-maze beginning at 30 days of age. The three lead-exposed groups (viz., maternal only, paternal only, or maternal plus paternal) made more errors in the maze than the controls, but did not differ from one another. However, offspring in the maternal plus paternal exposure group had longer swimming times than those in either maternally or paternally exposed groups, and those in the latter groups had longer swimming times than the controls. Thus, either maternal or paternal exposure exerted deleterious effects on the offspring, but maternal plus paternal exposure had the most severe effects. In another study, 90-day-old Sprague-Dawley rats were exposed for 70 days to 0.3% lead acetate in drinking water (blood lead of 58 pgidl ), or to 5 mg/cms; lead oxide via inhalation (aerosol) for 6 h/day, 5 days per week (blood lead of 51 kg/d]) (45). In addition to the standard reproductive endpoint assessments, these investigators mated the exposed males to unexposed females. Some of the mated females were sacrificed on gestation day 20 and examined for corpora lutea of pregnancy, implantation sites. resorptions, and number and sex ratio of fetuses (none of these parameters was affected by lead exposure). Other pregnant females were allowed to deliver and rear their litters, and the offspring were followed until they were 90 days old. At that time. some of the male off-
NELSON
ET AL.
spring were examined for reproductive parameters and the others were mated to determine fertility. The growth pattern of the exposed males did not differ from that of controls, and the only reproductive parameter affected was a reduction in the number of spermatozoa in the cauda epididymides. In spite of that reduction, there was no effect on the fertility of the offspring. Others have investigated male-mediated developmental disorders in rats (19). Paternal lead-induced changes have been observed in early gene expression in early embryos, as well as alterations in hippocampal development of late fetal and early neonatal rats. Taken together, these studies suggest the need for additional research on paternally mediated effects of lead-particularly because lead is an ubiquitous pollutant.
FEASIBILITY OF USING RABBITS TO DETECT MALE-MEDIATED EFFECTS
As described above, several chemicals have demonstrated paternally mediated developmental toxicity in experimental animals. The majority of experimental studies assessing function of offspring following maternal or paternal exposures have utilized rats as the experimental animal (21), but other species are used. For example, the National Toxicology Program (NTP) has initiated studies to validate the rabbit as an animal model for assessing male reproductive toxicity. The rabbit was chosen as the animal model because it is the smallest laboratory animal from which an ejaculate can be routinely collected. Ejaculated semen, assessing accessory sex gland secretions, more closely simulates human studies than does collection of epididymal sperm from rodent models. Being able to collect ejaculates allows for multiple samples. utilizing a longitudinal study design with experimental animals. An important part of reproductive toxicology is assessing the reproductive ability of males following exposure, as well as functional assessment of their offspring. One of the NTP validation studies is investigating the reproductive toxicology of lead administered to rabbits (3840,53,54,62,68). The merits of using rabbits in neurobehavioral testing have been described (25). A major point is that rabbits may provide an excellent model for behavioral teratology because maternal rabbits provide little care for their young-hence there is less concern about the need for fostering. Rabbits build a nest from available materials, including their own fur. However, once the babies are born, the doe leaves the pups and returns to nurse only once every 24 h. When she returns to nurse, the doe positions herself over the nest, not giving the pups any assistance in suckling. She does not brood them, clean them, or retrieve them to the nest. After lo-15 min of nursing, the doe departs until the next day. The neonates are born without fur, with eyes and outer ears sealed, and with poor motor coordination. Their eyes open around days 10-12. They develop thermoregulatory ability about day 15, around the time they start to leave the nest. By the time of weaning at around 5 weeks of age, their weight has increased from approximately 40 g at birth to 400-500 g. Given this information about young rabbits. the projected use of rabbits in male reproductive toxicology, and the availability of lead-exposed paternal rabbits, we conducted two exploratory studies. This article describes our attempt to use lead-exposed rabbits in a preliminary feasibility study to determine the utility of rabbit offspring for behavioral testing of offspring after paternal exposure. Based on the results of this preliminary study, the second phase consisted of the main
PATERNAL
EXPOSURE
OF RABBITS
study utilizing a range of doses numbers of exposed rabbits.
of lead,
193
TO LEAD along
with higher
METHOD
Details of the experimental design, dosing procedures, collection and analysis of blood and semen samples, histopathology of testes, and hormone analyses are presented separately for the preliminary studies (38,39,53,68) and the main studies (40,54,62). Briefly, in a project approved by our IACUC, sexually mature male and female Dutch Belted rabbits were purchased (6-7 months old; Hazelton Research Products; Aberdeen, MD). They were maintained in an AAALAC-approved facility in temperature-controlled rooms with tap water and Purina high-fiber rabbit chow (125 g/day) and a 12/12 light/ dark cycle. Lead acetate (O-3.85 mg/kg on Monday, Wednesday, and Friday) was administered SC to groups of rabbits (following 5 weeks of preexposure baseline acclimationitesting) for 15 weeks (through six cycles of the seminiferous epithelium to ensure that all sperm were formed under plateau lead exposure conditions). Weekly blood lead levels were measured by atomic absorption spectroscopy with Zeeman background corrections in a Centers for Disease Control and Prevention certified laboratory (a national reference laboratory). After the first 5 weeks of lead administration, the blood levels had reached a plateau, and were stable. In the preliminary phase, seven rabbits/group were given lead to reach target blood levels at Control (i 5), 50, and 110 kg/dl. In the main study, groups of 15 male rabbits/group were administered lead acetate sufficient to produce target blood levels at Control (< 5). 20,40, and 80 kgidl. In the preliminary study, males in the Control (actual mean = 0.2; limit of detection = l.l), 50 (actual mean = 5.5.9), and 110 (actual mean = 125.6) pg/dl blood lead levels were mated with virgin female rabbits within a week after the last lead injection. Because of the nature of the other testing involved with these males. all matings (3 groups X 7 rabbits per group = 21 rabbits) were accomplished in 2 days. The does were housed in 25 X 20 X 16-in. stainless steel cages and not disturbed, other than weekly weighing and cage cleaning, until expected parturition when litter boxes and nest material were provided. In the main study, males in the Control (actual mean = 0.6), 20 (actual mean = 24.6), 40 (actual mean = 38.9), and 80 (actual mean = 71.2) kg/d1 blood lead levels were mated with virgin female rabbits within a week after the last lead injection. To achieve minimal spread in birth of the young rabbits, yet still maintain the other desired testing, all 60 males (4 groups X 15 males) were mated over a period of 10 days. At the time of expected parturition, rabbits were checked each morning and evening for delivery. On the day of birth (day 0), the live offspring were counted. Otherwise, to minimize handling (and resulting probability of the mother killing the neonates), there was minimal contact with the pups (daily examination for number alive/dead, weekly cage changes). They were weighed individually on postnatal days 5,10, 15,20, 25,30, and (some on) 35. Testing was performed by personnel blind to the treatment groups of the rabbits being tested. Standard figure-eight rodent activity monitors (stainless steel, wire mesh flooring; Omnitech Electronics, Inc.; Columbus, OH) were used to assess exploratory motor activity (1). Pilot studies suggested that motor activity was minimal before the eyes were open (day 10-12). and peaked at around 25 days of age. By about 35 days of age, the rabbits’ size may have adversely affected their activity because of the size of the figure-eight corridors. Consequently, rabbits were tested on
days 15. 20, 25, and 30. Testing was for a 30-min test period, with data (number of entries into area of a new photocell, and total activity counts) recorded every 5 min. Another test that purportedly assesses exploratory activity involves the consumption of novel liquids [e.g., (22,35,65)], and this test was attempted in the preliminary study. This test also allowed us a simple test of learning (conditioned taste aversion). Patterned after the testing paradigm of Weinberg et al. (65), rabbits were tested on days 20-31. Days 20-25 served as the acquisition phase for the animals learning to consume the novel substance. On day 25, an aversive stimulus was administered to the rabbits following consumption of the novel liquid. Days 26-31 served as the recovery phase. From days 20-25, a 30% (w/w) sucrose solution was provided ad lib in bottles for 1 h at about 1200 h (acquisition phase; following the figure-eight testing on days 20 and 25). For this hour, littermates were placed together in a 22 X 12.5 X g-in. hanging polypropylene cage, and all fluid loss from the bottle was presumed to result from consumption by the young rabbits. After this test on day 25, each rabbit was injected IP with 7.5 ml/kg lithium chloride (LiCl; 0.4 M; Sigma Chemical Co.; St. Louis, MO), which serves as an aversive stimulus in animals (aversive conditioning phase). From days 26-31, rabbits were given the same liquid at approximately the same hours as from days 20-25 (recovery phase). (Because of the variability in response and lack of significant results, this test was not used in the main study.) The data were analyzed using multivariate analysis of variance (MANOVA) for repeated measures. In the preliminary study, even though a control and two dose levels of lead were included, loss of litters in the high-lead group rendered that group inappropriate to include in the statistical analyses (see the Results section). The null hypothesis (H,) in this test is that the means for all of the days under consideration, for a given dependent variable, are the same for all comparison groups. Rejection of H, implies that the means are different for at least 1 day. We used an F-test calculated from Wilk’s lambda as the criterion in our tests. We examined four dependent variables in the preliminary study. These included mean litter weight on days 5, 10, 15,20, 25, 30, and 35; mean litter “activity” and “entries” in the fig-
Offspring Weights - Preliminary Study 700 Legend
Jr
FIG. 1. Weight gain in offspring of rabbits paternally exposed
to lead (control, 50, and 100 kg/dl). Data from all three groups are presented, although excessive deaths in the high-lead group prohibited its inclusion in the statislical analysis (preliminary study).
194
NELSON
ET AL.
Sucrose Solution Consumed
Figure-8 Activity Chang.
by Agm (Acdvlty)
FIG. 3. Sucrose consumption in offspring exposed to lead (control, 50 t.qg/dl) (preliminary
of rabbits study).
paternally
300 Legend mo-
*~l.Y***.
n*b I..&
-----
Lowl...d
-
conbo(
low-lead, and control groups, respectively), with a mean number of six live births in each treatment group (range 2-8). Gestation length was not affected (30 -C 1 day). Significant postnatal deaths were observed in all groups, but were highest in the high-lead group. By 10 days of age, only two litters had surviving pups in the high lead group (two litters had no surviving pups, one litter had one pup, and one litter had six pups, for a total of seven pups); six litters had an average of 3.2 pups/litter in the low lead group (two litters had two pups,
M)-
0.8
summary activity data in offspring of rabbits paternally exposed to lead (control, 50, and 100 pg/dl). Data from all three groups arc presented, although excessive deaths in the high-lead
FIG.
2. Figure-eight
c~~~~~_~~_~~~~~~~~_~~~~~~~_
I-
group prohibited its inclusion in the statistical analysis. Top = number of entries. Bottom = total activity counts (preliminary study).
,’ V B
litter sucrose consumption for pairs of days 20 and 21,22 and 23,24 and 25, 26 and 27, 28 and 29, and 30 and 31 (this pairing was necessary to afford sufficient degrees of freedom to carry out the test). In the main study, ANOVA was used to test for day, period, and lead effects, and their interactions. The litter was the unit of analysis, with mean values for each litter calculated prior to analysis. MANOVA was used to test within-litter effects (day, period, and their interactions). An F-value calculated from Wilk’s lambda was used as the test statistic. Between-litter effects and contrasts were done with F-tests. Contrasts were done to compare lead values if there was a significant main effect or interaction involving lead. These calculations were done with SAS7 Version 6.11 (SAS Institute, Inc., Cary, NC) we-eight
monitors
on days
15,20,
25, and 30; and mean
RESULTS
_’
,
0.6
0.5
k? 1
0.4
a 0.3
0.2
0.1
0.0 0
5
10
15
26
25
80
Day of Age
Preliminary Study Of the 21 male rabbits that were mated in the preliminary study, 16 produced viable litters (4/7, 6/7, 6/7 in high-lead,
FIG. 4. Cumulative mean proportions dead of offspring of rabbits paternally exposed to lead (control, 20,40, and 80 kg/dl) as a function of age (main study).
PATERNAL
EXPOSURE
OF RABBITS
195
TO LEAD
DayofAge
-IS --20 ---
25
..-.-
30
400
22 E
*B 300 3
4
a
c
200
I
loo
5
x)
15
20
25
30
1
2
3
4
5
6
Period
FIG. 5. Weight gain (g) in offspring of rabbits paternally exposed lead (control,
20,40,
and 80 pgidl)
to
(main study).
DayofAge \
litter had three pups, one litter had five pups, one litter had seven pups, for a total of 19 pups); and six litters had an average of three pups/litter in the control group (one litter had two pups, one litter had three pups, two litters had four pups, and one litter had five pups, for a total of 18 pups). As discussed above, the high-lead group had only one litter with more than one pup per litter. Consequently, we did not include this group in the statistical analyses, but the data are included in the first two figures. Figure 1 presents the offspring weight gain through 3.5 days of age in the preliminary study, but no significant treatment-related effects were observed. Figure 2 presents the preliminary study figure-eight activity data, where there was evidence of possible differences between groups (Fig. 2A = number of entries [lambda = 0.21; F(4, 5) = 4.7, p = 0.061, Fig. 2B = total activity counts [lambda = 0.61; F(4,S) = 6.4, p = 0.031). Figure 3 presents the consumption of sucrose solution from days 2&31, where there was no evidence of group differences. one
‘\
\
\
\
\
-IS --20 --- 25 .----.---30
\ ‘1
Main Study
Of the 60 does mated in this study, 57 delivered litters (15/15, 15115, 13/15, and 14/15 in the control to high-lead group. respectively). Thus, the lead levels used in this study did not affect the reproductive ability of the male rabbits (mean implants per litter = 6.6. 7.6, 7.2, and 7.6 pups per litter, for the control to high-lead group, respectively). Gestation length was not affected (30 5 1 day). Two of the does died giving birth (one in the control group and one in the lowlead group). As in the preliminary study, nearly one half of the offspring were either dead at birth, or died within about 1 week after birth. Figure 4 shows the mean proportion of pups dead on days 0 to 30. The main effect of day was significant
1
2
4
3
5
6
Period FIG. 6. Overall mean period areas (top) and activity counts offspring (combined treatment and 30) (main study).
(5 min) by test age entries into new (bottom) of the figure-eight in rabbit groups) by day of age (day 15,20,25,
[lambda = 0.25; F(14,38) = 8.2, p = O.OOOl]. The main effect of lead approached significance (p = 0.07). Figure 5 shows the mean body weight of the pups as a function of day of age. The main effect of age was significant
NELSON
196 30
25 .-fi? : 20 w z ?I n 15 E ii! s
1
ET AL.
35
Lead (pg/dL)
I
CIO iQz 20 m 40 BZZ% 80
30 i y) 25 F 2 o 20 1 0 .z
10
r”
8
15-
5 =
IO-
5
0 15
Day
20
25
FIG. 7. Mean figure-eight “entries” (left) and “activity” (right) in offspring of rabbits paternally to lead (control. 20.40. and 80 kgidl) hy day ot’age (day 15.20.25. and 30) (main study).
[lambda = 0.03; F(S. 32) = 208.2. p = O.OOOl], but there were no treatment-related effects. Figure 6 shows the significant day X period interactions for entries [lambda = 0.21: F(lS, 21) = 5.2,~ = 0.0003] and activity [lambda = 0.33; F(15, 21) = 2.8. p = O.Ol]. As illustrated, young rabbits were most active during the first periods tested in the figure-eight at all ages. However. activity during the first half of the test was lowest on day 15, and highest on day 25. Figure 7 shows the significant lead X day interactions for entries [lambda = 0.59; F(9. X0) = 2.2, p = 0.031 and activity [lambda = 0.54: F(9. 80) = 2.6, 11 = O.Ol]. For entries: the mean (? SEM) of the 80 *g/d1 lead group (17.2 i 2.2) was significantly less than the mean of the 0 group (23.3 + 1.8) on day 25 (j = 0.03); the mean of the 40 kg/d1 lead group (16.2 5 1.7) was significantly less than the mean of the 0 group on day 25 (~9 = 0.01); and the mean of the 40 kg/d1 lead group (17.3 ? 1.7) was significantly greater than the mean of the 20 pgidl lead group (11.9 -t 1.7) on day 30 (JI = 0.03). For activity: the mean of the 80 kg/d1 lead group (23.5 ? 2.8) was nearly significantly less than the mean of the 0 group (30.6 5 2.3) on day 25 (p = 0.05): the mean of the 40 pgidl lead group (21.4 5 2.2) was significantly less than the mean of the 0 group on day 25 (p = 0.005): and the mean of the 40 kg/d1 lead group was significantly less than the mean of the 20 p.g/dl lead group (28.8 ? 2.2) on day 25 b = 0.02). DISCIJSSION
The data in the highest lead group (110 pgidl blood lead. preliminary study) are suggestive of an adverse effect of lead on fertility (417 litters). However, no effect on fertility was noted at 75-80 pg/dl blood lead, and additional research will be needed to verify the antifertility effects at the highest level. Additional research will also be necessary to determine if the incidence of postnatal deaths in rabbits in the high-lead group (110 pgidl blood lead. preliminary study) was treatment related, as the sample sizes were too small to make definitive statements. The next lower dose (75-80 kg/d1 blood lead) did not appear to produce effects on postnatal deaths. (Although the incidence of postnatal deaths seems high, con-
30
35
Day exposed
versations with laboratory rabbit breeders indicate that this rate is not unusual for prima gravida females.) Our data suggest that the levels of lead we used do not affect offspring weight gain, at least through the time of weaning. This lack of effects on fertility or on weight gain is consistent with the lack of effects seen in male rats after paternal lead exposure (45). The pattern of activity seen in the young rabbits (viz. an increase in activity to a peak and then a decrease in activity with age) is typical of that seen in rats, with the peak somewhat dependent on the activity measuring device [e.g., (l)]. It appears that the peak we observed was a real peak observed in behavior (the consistency seen in three “replications,” viz., a few pilot litters, those in the preliminary study. as well as those in the main study, is quite striking), although it is possible that the size of the rabbits may have affected their activity at the oldest age tested. The pattern of high activity during the first period of test, decreasing to little or no activity by the end of testing (Fig. 6). is typical of that reported in figure-eight testing [e.g., (9)]. In fact, the lJS EPA guideline on motor activity assessments specifies that the duration of testing allow motor activity to approach asymptotic levels by the last 20% of the session (63). The hypoactivity we observed after paternal lead exposure (day 25: reflected both in the number of entries into new areas as well as in the overall activity: Fig. 7) is similar to that reported after maternal lead exposure [e.g., (46)], although hyperactivity and no effects on activity have also been reported (1 I). As the latter investigators point out, different measures of activity do not necessarily correlate with one another. Furthermore. activity levels can be influenced by species, age, sex, estrous cycle, time of day, novelty of environment. experience. and food deprivation. etc. We did not find literature references to consumption of sucrose or the use of lithium chloride in rabbits. The 30% sucrose solution and dose of lithium chloride we selected were based upon references using rats. If rabbits are to be used extensively for this kind of research, more detailed investigations should assess the appropriateness of the sucrose and lithium chloride levels we used. Extensive research has been conducted with neonatal rabbits. particularly investigating the development of olfactory
PATERNAL
EXPOSURE
OF RABBITS
TO LEAD
197
function and odor conditioning [e.g., (23-27,56)]. Because of the more extensive testing that would be involved, and the clustering of births in our study, we did not investigate olfactory function. However, utilization of the data and experience of Hudson and Distel would add additional testing capabilities to those we used. A rabbit model for studying the prenatal effects of cocaine has recently been described (41) along with conditioning and discrimination learning (49-51). In summary, these data suggest the need for more research into paternally mediated effects, in experimental animals as well as in humans. Based on the present study, we believe that rabbits can be used to detect postnatal functional deficits following paternal exposures to exogenous agents. However, the high rate of postnatal deaths in rabbits, as opposed to that seen in rats, makes the use of rabbits less favorable for routine screening than rats. Nonetheless, when rabbits would make
better models for detecting the adverse reproductive effects of the specific chemical or physical agents being tested, rabbits can be used for the postnatal studies as well. Our data suggest no effects of paternal exposure below 75-80 kg/d1 blood lead on fertility, survivability of offspring, or weight gain through weaning in rabbits. It does appear, however, that exploratory activity may be affected in the offspring at and above 40 kg/d1 blood lead.
ACKNOWLEDGEMENTS We are grateful for the support of Dr. Bob Chapin and NIEHSI NTP throughout these studies (Interagency Agreement #YOl-ES40266 “Validation of the Rabbit Model for Assessing Reproductive Toxicants”). We thank the animal caretaker staff, particularly Alissa Christman. for their excellent care of the young rabbits in the studies.
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