FUNDAMENTAL
AND
APPLIED
TOXICOLOGY
19,4
1l-422 ( 1992)
Gonadal Effects of Fetal Exposure to the Azo Dye Congo Red in Mice: Infertility in Female but Not Male Offspring’ L. E. GUY, Developmental
JR., J. S. OSTBY, R. J. KAVLOCK,
Reproductive and *ManTech
Biology Section, Reproductive Inc., Research Triangle Park,
AND R. MARSHALL*
Toxicology Branch/HERL/USEPA, North Carolina 27711
Received September 19, 199 1; accepted April 9, 1992
(NIOSH, 1980). Benzidine causes cancer of the bladder in humans (Haley, 1975) and its congeners are known to be animal carcinogens (NIOSH, 1980). In a 13-week subchronic study conducted at the National Cancer Institute, a significant excess of hepatocellular carcinomas and hepatic neoThe present study describes the relationship between gonadal plastic nodules was demonstrated in rats (NCI, 1978). The agenesis and fertility in male and female mice exposed in utero time to tumor for the carcinomas was the shortest ever reto the diazo dye Congo red (CR). Maternal CR treatment in- ported in the Institute’s Bioassay Program (NIOSH, 1980). hibited testicular and ovarian function in the offspring after oral In recognition of the adverse human health effects of expoadministration of 1 or 0.5 g/kg/day on Gestational Days 8-12. sure to benzidine-based dyes, NIOSH issued a Special Hazard The testes of male offspring from CR-exposed dams were small in size and contained hypospermatogenic seminiferous tubules. Review, which concluded that exposure to the dyes should However, despite the fact that testis weight wasreduced by more be eliminated (NIOSH, 1980). In addition to their carcinogenic and mutagenic potential than 70% in some males, they displayed normal levels of fertility (Reid et al., 1984), some of the azo dyes, including trypan when mated to untreated females for over 10 months. In contrast, female offspring from CR-exposed dams produced only about blue and Congo red (Beaudoin, 1964; Beaudoin and Pickhalf as many litters and pups as the control pairs did under long- ering, 1960; Lloyd and Beck, 1966; Wilson, 1955; Beck and term mating conditions. Histological examination of the ovaries Lloyd, 1966) have embryopathic effects in rodents. The azo revealed that subfertility was correlated with ovarian atrophy. dye trypan blue is a classic teratogenic agent that has been Females lacking maturing follicles were considerably less pro- extensively studied over the past 35 years. After maternal ductive (1.3 litters and 11.5 pups) than treated females with histologically normal ovaries (7.1 litters and 78.1 pups). In sum- injection, trypan blue accumulates in the endodermal cells mary, prenatal exposure to the dye CR affects the gonads of of the visceral yolk sac, leading to disturbances in growth both male and female offspring, but only the female offspring and differentiation of the embryo (Beck, 198 I). It has been postulated that the teratogenic effect of trypan blue is due display reduced fertility. o 1992Society of Toxicology. to disruption of the nutritive function of the yolk sac placenta. In contrast to the numerous studies of the developmental Azo dyes based on benzidine and the dimethyl- and dimethoxybenzidine congeners constitute a large group of toxicity of the diazo dyes after maternal injection, the developmental effects of oral exposure to these dyes have not chemicals that have been widely used in the textile, leather, been determined. Such studies were not considered because and paper industries. Several hundred dyes are derived from these chemicals. In 1978, domestic production and imports the azo dyes are unlikely to reach the fetus after maternal/ amounted to about 4.1 million pounds of these dyes, with oral administration, being poorly absorbed in the gut. Howtens of thousands of workers being occupationally exposed ever, benzidine and the other parent amines are formed from (NIOSH, 1980). Studies from as early as 1895 reported that these dyes by microbial metabolism in the intestinal tract. workers in the dye industry have an incidence of urinary These metabolites, unlike the dyes themselves, are readily bladder tumors greater than that in the general population absorbed in the gut (Bos et al., 1986; Bowman et al., 1982, 1983) and subsequently appear in the urine of humans ’ This report has been reviewed by the EPA’s Office of Research and (Lowry et al., 1980) monkeys (Rinde and Troll, 1975) roDevelopment and approved for publication. Approval does not signify that dents, and dogs (Lynn et al., 1980). Hence, it is likely that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or benzidine would be available to the fetus after maternal CR administration. In fact, studies in our laboratory indicate recommendation for use. Gonadal Effects of Fetal Exposure to the Azo Dye Congo Red in Mice: Infertility in Female but Not Male Offspring. GRAY, L. E., JR., OSTBY, J. S., KAVLOCK, R. J., AND MARSHALL, R. (1992). Fundam. Appl. Toxicol. 19,411-422.
411
0272-0590/92 Copyright All rights
$5.00
0 1992 by the Society of Toxicology. of reproduction in any form reserved.
GRAY ET AL.
412
TABLE 1 Summary of the Experimental Protocols of the Three Experiments in the Current Study Experiment No. Dose
Objective Methods
Experiment 1 1 g/k/day
Determination of effect of congo red on fertility Male and females from the same treatment group bred continuously to 300 days of age; testes weight
Experiment 2a 1 g/kg/day
Determination of fertility effect by sex Continuous breeding: experimental male with two control females to Day 360 and an experimental female with a control male to Day 270
Experiment 2b 1 g/kg/day
Description of age-related expression of toxicity Housed in unisexual groups Necropsied at 90, 170, or 320 days of age In 2a and 2b: testis weight and histology and sperm concentration in the vas deferens in males and ovarian histology in females
Experiment 3
Description of the effects of Congo red on sperm numbers, testis weight, and histology Testis, cauda epididymal, and seminal vesicle weights, caudal sperm reserves, and testicular histology at Days 47 and 185; testicular sonication-resistant sperm head count on Day 185
0.5 mg/kg
that Congo red causes developmental reproductive toxicity when the mother is dosed by gavage during the period of major organogenesis (Gray et al., 1989a; Gray and Kavlock, 1984). We found that administration of CR to pregnant mice and rats by gavage (1 g/kg/day on Days 8- 12) reduced tes-
titular weight in the offspring. In contrast to CR, we found that a number of structurally unrelated dyes failed to alter murine testicular development. Initially, similarly treated male and female mice were paired continuously to assessfertility. When subfertility was detected, CR-treated offspring were mated long term with untreated mice to determine the affected sex in a second experiment. Our continuous breeding protocol is modeled after the “Forced Breeding Protocol” employed by McLachlan et al. (1982), which they employed to detect subfertility in mice after prenatal exposure to diethylstilbestrol (DES). Thus, this study was designed to (1) relate the fertility of male and female, determined in a continuous breeding protocol, to the gonadal alterations; (2) to determine the affected sex, if infertility was observed, (3) determine if the gonadal alterations changed as the animals aged; and (4) compare the alterations in testis weight to other potentially more sensitive measures, including testicular sperm production, seminiferous tubular atrophy, and cauda epididymal sperm reserves. METHODS Primiparous 90-day-old CD-l mice were received from Charles River Breeding Laboratory (Wilmington, MA) on Day 5 of gestation. The mice were individually housed in clear plastic cages(33 X 20 X 13 cm) with heattreated laboratory-grade pine shavings as litter, maintained on a 12: 12 photoperiod at 20-24°C (relative humidity, 40-50’S), and given Wayne Lab Blox and tap water ad libitum. Dams were weighed, weight ranked, assigned to treatment on Day 7 of gestation, dosed on Days 8- 12, and weighed again on Day 13. Pups were counted and weighed during the neonatal period. Dams that had not delivered by Postnatal Day 3 were killed using COr to determine if they were not pregnant or had resorbed their litter. A summary
TABLE 2 Maternal and Neonatal Effects of Gestational Administration Experiment No.: Treatment:
of Congo Red in Experiments 1 and 2
1 Con
2 CR oral
Con
CR oral
25 0 9 1 15
15 1 5 1 8
36 2 3 0 31
17 0 4 0 13
7.4 + 0.70”
6.0 + 0.67
8.5 + 0.21
9.0 ?I 0.73
9.8 f 0.3 1.66 + 0.02
10.8 f 0.4 1.61 ? 0.03
Maternal data No. No. No. No. No.
dosed late not pregnant dead delivering on time
Maternal weight gain during treatment (g)
Neonatal data (Day 1) No. Live pups Pup weight (g)
7.9 k 1.2 1.66 + 0.05
8.4 r 0.9 1.60 + 0.07
Note. Con, control; CR, Congo red. The dye was administered orally at 1 g/kg/day on Days 8-12 of gestation. There were no significant treatment effectson any maternal or neonatal parameters. ’ Values are means f standard errors.
EFFECTS OF FETAL
Paired Testes
EXPOSURE
Number of Litters
TO CONGO
Per Breeding
RED
413
Produced
Pair
320 -
14-
140-
300 -
13-
130-
280 -
12-
120-
260 -
11 -
llO-
240
lo-
loo-
-
220 -
9-
90 -
200 -
8-
8-
80 -
180-
7-
7-
70 -
160-
6-
6-
60 -
140’
5-
5-
50 -
120-
4-
4-
40 -
loo-
3-
3-
30 -
2-
20 -
l-
lo-
80 -
2-
60 -
l-
t. .. IIl 1 J 1 1 J l mmm
-
Control
Congo
Males
Red
-
Control
Congo
Breeding
Per
Red
0
Control
Congo
Red
0
Contrc
Pairs
FIG. 1. The effects of oral administration of Congo red at 1 g/kg/day to the dam on Days 8- 12 of gestation on the reproductive physiology of the offspring. Data are from Experiment 1. Breeding pairs were established at 35 days of age using a male and a female from the same treatment groups. Data were collected up to 300 days of age. ( “:” denotes p < 0.05 different from control). The data analysis for “Mean litter size per breeding pair” did not include the four litters that did not breed, but the data from these pairs were included for other measures shown in this figure.
of the experimental designs and general methods employed in the present study is shown in Table 1. In the first experiment, the dams (PO generation) were treated with CR at 1 g/kg/day in water on Days 8-12 (Day 0 is the day of mating) by gavage and allowed to litter (the Fl). The numbers of dams in each group are shown in Table 2. Dams that had not delivered their litters within 24 hr of the expected delivery day were classified as “late.” On Postnatal Day 3, 8 control dams were randomly selected and assigned 3 pups/sex, randomly selected from the population of control pups. Six CR-treated litters were similarly established. On Postnatal Day 35, 14 control and 16 CR Fl breeding pairs were established. Both members of each breeding pair were from the same treatment group. Birth of the F2 pups was monitored over a 9-month period, during which the date of birth of the F2 pups and number of pups per litter were determined shortly after birth. At this time the F2 were removed and euthanized. At 300 days of age the Fl males were killed with carbon dioxide and necropsied and the females were euthanized. In males, testes, seminal vesicle, liver, kidney, and body weights were measured. A second experiment was designed to determine: (1) if a female factor was responsible for the infertility seen in the Fl CR-treated breeding pairs in the first study, (2) if the marked reduction in testis weight following prenatal CR treatment produced infertility, and (3) if the gonadal effects were reversible or became more severe with age. In this experiment, as in the first, the PO dams were dosed orally with CR at 1 g/kg/day or the vehicle (water) on Days 8-12 of gestation (sample sizes are shown in Table 2). At weaning, Fl mice were randomly assigned to breeding
pairs or unisexual housing conditions. For the Fl breeding component, 20 control and 19 CR-treated males were placed in breeding cages with 2 90-day-old untreated females/male. Pregnant females were allowed to deliver, pups (F2) were counted, litters were removed, and the female was replaced. These conditions were maintained until the males were necropsied when they were 360 days old. In order to assessthe fertility of the female offspring, 20 control and 20 CR-treated females were each paired with an untreated 90-day-old male. The males were replaced twice, when the females were 90 and 190 days of age. Fl females were necropsied at 9 months of age. In the second experiment, unisexual groups were also established in order to examine age-related changes in gonadal effectsin male and female mice. These groups consisted of 3 males/cage or 5 females/cage, with sample sizes of 45 CR males, 45 control males, 45 control females, and 30 CR females. At 90, 170, and 320 days of age approximately a third of the animals from each group were necropsied. In Experiment 2, body, testis, seminal vesicle, liver, and kidney weights were measured, a small sample of fluid from the vas deferens was taken, and sperm concentration was determined. One side of the reproductive tract, including the testis and epididymis, was fixed in Bouin’s, rinsed with alcohol, embedded in parathn, stained with H and E, and evaluated microscopically (Experimental Pathology Lab, NC). Testes were also examined histologically using a projecting microscope and the numbers of normal and completely atrophic tubules per cross section of testis were counted. In females, body weight was recorded at necropsy and the ovaries were fixed, as above, and examined for histopathological lesions.
414
GRAY ET AL.
TABLE 3 Maternal and Pup Data for Experiment 3
Con No. of litters Dam’s weight gain from Days 8 to 12 of gestation (g) % postimplantation loss
12
No. pups/litter on Day 1 Average pup weight on Day 1 k) Pup weight gain on Days 1-6 (g) % mortality on Days l-6 Pup weight gain on Days 6-29 % mortality on Days 6-29
11.7
(z&
(1.2) 1.66 (0.05) 2.11 (0.13) 0.03 (0.01) 36.2 (0.8) 6.0 (0.02)
CR (0.5 mg/kg/day) 6 4.3 (0.5) 10.8 (5.4) 10.0 (1.1) 1.I3 (0.04) 2.35 (0.15) 0.00 (0.00) 34.8 (0.7) 6.0 (0.00)
Note. Dams were dosed with 0.5 g/kg/day on Days 8-12 ofgestation with the azo dye CR or the vehicle (0.2 ml of water) (Con). There were no significant alterations of any of the maternal or neonatal measures. ’ Values are means (standard error of the mean).
A third experiment was designed with three objectives in mind: (1) to determine if a lower dose of CR affectedthe testis of the male fetus (CR was administered at 0.5 mg/k&day in 0.2 ml of water on Days 8-12 of gestation); (2) to compare the sensitivity of other indices of testicular function to testis weight; and (3) to determine if litter effectswere present in the data set. Pups were kept with their natural mothers, litters were randomly reduced (not culled) to seven pups (six males and one female, whenever possible), and on Postnatal Days 1, 6, and 29 the pups were weighed. The pups were weaned on Day 30, the dams were killed, and the numbers of implants were counted. Males from the third study were necropsied at 46-47 and 187- 190 days of age, at which time the left side of the reproductive tract was used for histopathology and body, testis, seminal vesicle, and cauda epididymal weights were recorded. In addition, cauda epididymal sperm reserves and sonication-resistant testicular sperm head counts were measured (see Gray et al, 1989b. for detailed methods). The females were not studied in this experiment. The preweanling data in the three experiments were analyzed using PRDC GLM, a general linear models procedure available on the SAS (SAS Institute, Inc., 1985), with the litter as the unit of statistical analysis. In Experiments 1 and 2. the postweanling data were analyzed as a completely randomized design because pups were not reared with their natural mothers and, hence, litter effectsare pooled with the general error term. In the third experiment, the postweanling data were analyzed as a nested analysis of variance, because pups were reared with littermates. This analysis removes the litter effect from the overall error term and the error variance is partitioned into a between-litter error variance (based on the variability between litters) and a within-litter error term (based on variability between littermates within litters). The treatment effect is a between-litter variable. The data from these experiments were also analyzed using nonparametric analyses (Fisher’s exact test). This was done because a comparison of the distributions of the testis weight data of control and CR males indicated that CR treatment increased
the variance and skewed the distribution of the data. In such situations, nonparametric analyses may provide a more accurate description of the data and be more sensitive. In this analysis, individual testis weights were compared against the mean value of the concurrent control group and those organs which weighed less than the control means minus three standard deviations were classified as abnormal. We have found that a testis that falls into this weight category typically contains atrophic tubules. Subsequently,
TABLE 4 The Effects of Prenatal Dye (Congo Red) Administration (1 g/kg/day, Days 8-12 of Gestation, by Gavage) on the Organ Weights, Seminiferous Tubular Atrophy, and Sperm Concentration in the Vas Deferens of Male Mice at Days 90, 170, 320, and 360 (Breeding Pairs) for Experiments 2a and 2b Days of age 90
170
320
360
Pooled
18 21
19 14
62 59
40.5 40.5
43.0 43.8
No. of males Control Congo red
12 12
13 12
Body weight (g) Control Congo red
39.9 40.7
43.5 44.3
46.9 41.3
Pooled SD, 3.67; treatment effect, NS; age, p < 0.0001; interaction, NS Testis weight (mg) Control Congo red
137 88
142 94
140 96
143 95
141 94
Pooled SD, 36.5; treatment effect, p < 0.0001; age, NS; interaction, NS No. of atrophic tubules/testicular cross section Control Congo red
1.1 13.2
0.8 21.2
0.8 18.2
3.6 14.1
1.7 16.8
Pooled SD, 13.1; treatment effect, p < 0.000 1; age, NS; interaction, NS No. of tubules/testicular cross section Control Congo red
316 227
270 217
274 227
283 226
284 225
Pooled SD, 52.4; treatment effect, p < 0.0001; age, NS; interaction, NS Vas deferens sperm concentration ( IO6 sperm/mg fluid) Control Congo red
1.84 1.38
1.70 1.18
1.76 1.14
1.24 1.04
1.60 1.17
Pooled SD, 0.68; treatment effect, p i 0.0008; age, 0.053; interaction, NS Seminal vesicle weight (mg) Control Congo red
415 389
524 527
754 642
587 551
587 547
Pooled SD, 132; treatment effect, NS; age, p c 0.000 1; interaction, NS Nore. SD, standard deviation; NS, not significant.
EFFECTS OF FETAL
EXPOSURE
TO CONGO RED
E‘IG. 2. Prenatal maternal administration of the azo dye Congo red (1 mg/kg/day, Days 8-12 of gestation) produces mild to severe testicular age due to hypospermatogenesis in affected seminiferous tubules in male mice. A normal testis is shown at the top, while the testis of a mildly affected CRbsed mouse is shown at the bottom (X 160).
GRAY ET AL.
416
the numbers of normal and abnormal testes in each treatment group were compared against the concurrent control using a Fisher’s exact test.
TABLE 5
Summary of the Nonparametrlc Analyses of the Data on Testis Weight and the Number of Tubules per Testicular Cross Section without Spermatogenic Cells
Maternal
Congo red
Control
Classification based on the number of atrophic tubules Experiment 2 No. of normal males No. of abnormal males Experiment 3 No. of normal males No. of abnormal males
RESULTS
60
1
39 20
31 0
9 9
p = 1.25
CR treatment did not adversely affect the viability or weight of the dams or their pups in any of the three experiments (Tables 2 and 3). Puberty (vaginal opening) was not delayed in CR-treated female offspring (data not shown). X 10m6 Male Necropsy Data x 1O-5
p = 2.4
In male offspring from the first experiment, testes weights were reduced by prenatal CR treatment (Fig. l), but body, seminal vesicle, liver, and kidney weights did not differ from control values, and no gross malformations were noted (data not shown). Similar effects were noted in the second and third experiments. The necropsy data from males housed in unisexual groups and killed at 90, 170, and 320 days of age and from the breeding males, killed at 360 days of age, from the second experiment are shown in Table 4. Body weight and seminal vesicle (with fluid) weight varied with age, but were not affected by CR treatment, and there were no interactions between these main effects. In marked contrast to body and seminal vesicle weights, testis weights did not change with age and they were consistently reduced by CR treatment at all ages (pooled means are 14 1 mg for control vs 94 for CR males). An example of a mild form of the testicular lesion seen in the CR-exposed males is shown in Fig. 2. In this experiment, the severity of hypospermatogen-
Classification based on testicular weight Experiment 1 No. of normal males No. of abnormal males Experiment 2 No. of normal males No. of abnormal males Experiment 3 No. of normal males No. of abnormal males
14 0
5
10 p = 1.5 x lo-5
62
34 25
0 31
p = 1o-8
4
14 p = 10-E
0
Note. A plot of the frequency distribution of the controls from all of our studies indicated that 98.5% of these males had fewer than 20 atrophic tubules per cross section. For this reason, testes with more than 20 completely atrophic tubules were classified as abnormal for the current analyses. Testes weights that were three standard deviations below the concurrent control mean were classified as abnormal.
22
I
I
I
I
I
and Neonatal Parameters
I
I
I
I
I
I
I
I
I
20 -
I
18-
I I -
3 c 0 z g @ '= 3 5
‘Severe’
Tubular
Congo
Red: 29%
0-q i
:
I
-++
\\
I
I
I
I
I
Control Males Congo Red Males
-
1614-
Control: 99% Congo Red: 52%
12lot. 61 4-
0
10
20
30
40
50
60
70
60
90
100110
120130140150160170160
Right Testis Weight,
190200210
(mg)
FIG. 3. A comparison of the frequency distributions of the testis weight and histology data from CR-exposed and untreated male mice from Experiments 2A and 2B. Pregnant females were dosed with the vehicle or the dye at 1 g/kg/day on Days S- 12 of gestation and their male offspring were necropsied at 90, 170, 320, or 360 days of age. The control and treated values were pooled across age because testis weights and the degree of hypospermatogenesis did not change from 90 to 360 days.
EFFECTS OF FETAL
EXPOSURE
TABLE 6 Male Offspring Necropsy Data for Experiment 3
Total No. of litters No. animals on Day 46 No. animals on Day 187 Body Weight (g) Day 46 Day 187 Pooled Testis weight (mg) Day 46 Day 187 Pooled SV weight (mg) Day 46 Day 187 Pooled Cauda epididymal weight (mg) Day 46 Day 187 Pooled Cauda epididymal sperm count (X106) Day 46 Day 187 Pooled No. of tubules per cross section of the testis Day 46 Day 187 Pooled No. of atrophic tubules per cross section Day 46 Day 187 Pooled Testicular sperm head count (X 106) Day 187
Con
CR
8 16 15
6 12 6
30.8 51.3 41.1
31.9 47.1 39.5
112 145 128
64 62 63
113 421 267
126 412 269
10.1 18.5 14.3 1.15 7.11 4.03 258 252 255 0.4 0.0 0.2
8.9 18.5 13.7 0.53 5.63 2.44 189 149 168 32.6 23.0 27.8
SDIEp
9.5/1.8, NS
33/42.1,
AND
APPLIED
TOXICOLOGY
19,4
1l-422 ( 1992)
Gonadal Effects of Fetal Exposure to the Azo Dye Congo Red in Mice: Infertility in Female but Not Male Offspring’ L. E. GUY, Developmental
JR., J. S. OSTBY, R. J. KAVLOCK,
Reproductive and *ManTech
Biology Section, Reproductive Inc., Research Triangle Park,
AND R. MARSHALL*
Toxicology Branch/HERL/USEPA, North Carolina 27711
Received September 19, 199 1; accepted April 9, 1992
(NIOSH, 1980). Benzidine causes cancer of the bladder in humans (Haley, 1975) and its congeners are known to be animal carcinogens (NIOSH, 1980). In a 13-week subchronic study conducted at the National Cancer Institute, a significant excess of hepatocellular carcinomas and hepatic neoThe present study describes the relationship between gonadal plastic nodules was demonstrated in rats (NCI, 1978). The agenesis and fertility in male and female mice exposed in utero time to tumor for the carcinomas was the shortest ever reto the diazo dye Congo red (CR). Maternal CR treatment in- ported in the Institute’s Bioassay Program (NIOSH, 1980). hibited testicular and ovarian function in the offspring after oral In recognition of the adverse human health effects of expoadministration of 1 or 0.5 g/kg/day on Gestational Days 8-12. sure to benzidine-based dyes, NIOSH issued a Special Hazard The testes of male offspring from CR-exposed dams were small in size and contained hypospermatogenic seminiferous tubules. Review, which concluded that exposure to the dyes should However, despite the fact that testis weight wasreduced by more be eliminated (NIOSH, 1980). In addition to their carcinogenic and mutagenic potential than 70% in some males, they displayed normal levels of fertility (Reid et al., 1984), some of the azo dyes, including trypan when mated to untreated females for over 10 months. In contrast, female offspring from CR-exposed dams produced only about blue and Congo red (Beaudoin, 1964; Beaudoin and Pickhalf as many litters and pups as the control pairs did under long- ering, 1960; Lloyd and Beck, 1966; Wilson, 1955; Beck and term mating conditions. Histological examination of the ovaries Lloyd, 1966) have embryopathic effects in rodents. The azo revealed that subfertility was correlated with ovarian atrophy. dye trypan blue is a classic teratogenic agent that has been Females lacking maturing follicles were considerably less pro- extensively studied over the past 35 years. After maternal ductive (1.3 litters and 11.5 pups) than treated females with histologically normal ovaries (7.1 litters and 78.1 pups). In sum- injection, trypan blue accumulates in the endodermal cells mary, prenatal exposure to the dye CR affects the gonads of of the visceral yolk sac, leading to disturbances in growth both male and female offspring, but only the female offspring and differentiation of the embryo (Beck, 198 I). It has been postulated that the teratogenic effect of trypan blue is due display reduced fertility. o 1992Society of Toxicology. to disruption of the nutritive function of the yolk sac placenta. In contrast to the numerous studies of the developmental Azo dyes based on benzidine and the dimethyl- and dimethoxybenzidine congeners constitute a large group of toxicity of the diazo dyes after maternal injection, the developmental effects of oral exposure to these dyes have not chemicals that have been widely used in the textile, leather, been determined. Such studies were not considered because and paper industries. Several hundred dyes are derived from these chemicals. In 1978, domestic production and imports the azo dyes are unlikely to reach the fetus after maternal/ amounted to about 4.1 million pounds of these dyes, with oral administration, being poorly absorbed in the gut. Howtens of thousands of workers being occupationally exposed ever, benzidine and the other parent amines are formed from (NIOSH, 1980). Studies from as early as 1895 reported that these dyes by microbial metabolism in the intestinal tract. workers in the dye industry have an incidence of urinary These metabolites, unlike the dyes themselves, are readily bladder tumors greater than that in the general population absorbed in the gut (Bos et al., 1986; Bowman et al., 1982, 1983) and subsequently appear in the urine of humans ’ This report has been reviewed by the EPA’s Office of Research and (Lowry et al., 1980) monkeys (Rinde and Troll, 1975) roDevelopment and approved for publication. Approval does not signify that dents, and dogs (Lynn et al., 1980). Hence, it is likely that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or benzidine would be available to the fetus after maternal CR administration. In fact, studies in our laboratory indicate recommendation for use. Gonadal Effects of Fetal Exposure to the Azo Dye Congo Red in Mice: Infertility in Female but Not Male Offspring. GRAY, L. E., JR., OSTBY, J. S., KAVLOCK, R. J., AND MARSHALL, R. (1992). Fundam. Appl. Toxicol. 19,411-422.
411
0272-0590/92 Copyright All rights
$5.00
0 1992 by the Society of Toxicology. of reproduction in any form reserved.
GRAY ET AL.
412
TABLE 1 Summary of the Experimental Protocols of the Three Experiments in the Current Study Experiment No. Dose
Objective Methods
Experiment 1 1 g/k/day
Determination of effect of congo red on fertility Male and females from the same treatment group bred continuously to 300 days of age; testes weight
Experiment 2a 1 g/kg/day
Determination of fertility effect by sex Continuous breeding: experimental male with two control females to Day 360 and an experimental female with a control male to Day 270
Experiment 2b 1 g/kg/day
Description of age-related expression of toxicity Housed in unisexual groups Necropsied at 90, 170, or 320 days of age In 2a and 2b: testis weight and histology and sperm concentration in the vas deferens in males and ovarian histology in females
Experiment 3
Description of the effects of Congo red on sperm numbers, testis weight, and histology Testis, cauda epididymal, and seminal vesicle weights, caudal sperm reserves, and testicular histology at Days 47 and 185; testicular sonication-resistant sperm head count on Day 185
0.5 mg/kg
that Congo red causes developmental reproductive toxicity when the mother is dosed by gavage during the period of major organogenesis (Gray et al., 1989a; Gray and Kavlock, 1984). We found that administration of CR to pregnant mice and rats by gavage (1 g/kg/day on Days 8- 12) reduced tes-
titular weight in the offspring. In contrast to CR, we found that a number of structurally unrelated dyes failed to alter murine testicular development. Initially, similarly treated male and female mice were paired continuously to assessfertility. When subfertility was detected, CR-treated offspring were mated long term with untreated mice to determine the affected sex in a second experiment. Our continuous breeding protocol is modeled after the “Forced Breeding Protocol” employed by McLachlan et al. (1982), which they employed to detect subfertility in mice after prenatal exposure to diethylstilbestrol (DES). Thus, this study was designed to (1) relate the fertility of male and female, determined in a continuous breeding protocol, to the gonadal alterations; (2) to determine the affected sex, if infertility was observed, (3) determine if the gonadal alterations changed as the animals aged; and (4) compare the alterations in testis weight to other potentially more sensitive measures, including testicular sperm production, seminiferous tubular atrophy, and cauda epididymal sperm reserves. METHODS Primiparous 90-day-old CD-l mice were received from Charles River Breeding Laboratory (Wilmington, MA) on Day 5 of gestation. The mice were individually housed in clear plastic cages(33 X 20 X 13 cm) with heattreated laboratory-grade pine shavings as litter, maintained on a 12: 12 photoperiod at 20-24°C (relative humidity, 40-50’S), and given Wayne Lab Blox and tap water ad libitum. Dams were weighed, weight ranked, assigned to treatment on Day 7 of gestation, dosed on Days 8- 12, and weighed again on Day 13. Pups were counted and weighed during the neonatal period. Dams that had not delivered by Postnatal Day 3 were killed using COr to determine if they were not pregnant or had resorbed their litter. A summary
TABLE 2 Maternal and Neonatal Effects of Gestational Administration Experiment No.: Treatment:
of Congo Red in Experiments 1 and 2
1 Con
2 CR oral
Con
CR oral
25 0 9 1 15
15 1 5 1 8
36 2 3 0 31
17 0 4 0 13
7.4 + 0.70”
6.0 + 0.67
8.5 + 0.21
9.0 ?I 0.73
9.8 f 0.3 1.66 + 0.02
10.8 f 0.4 1.61 ? 0.03
Maternal data No. No. No. No. No.
dosed late not pregnant dead delivering on time
Maternal weight gain during treatment (g)
Neonatal data (Day 1) No. Live pups Pup weight (g)
7.9 k 1.2 1.66 + 0.05
8.4 r 0.9 1.60 + 0.07
Note. Con, control; CR, Congo red. The dye was administered orally at 1 g/kg/day on Days 8-12 of gestation. There were no significant treatment effectson any maternal or neonatal parameters. ’ Values are means f standard errors.
EFFECTS OF FETAL
Paired Testes
EXPOSURE
Number of Litters
TO CONGO
Per Breeding
RED
413
Produced
Pair
320 -
14-
140-
300 -
13-
130-
280 -
12-
120-
260 -
11 -
llO-
240
lo-
loo-
-
220 -
9-
90 -
200 -
8-
8-
80 -
180-
7-
7-
70 -
160-
6-
6-
60 -
140’
5-
5-
50 -
120-
4-
4-
40 -
loo-
3-
3-
30 -
2-
20 -
l-
lo-
80 -
2-
60 -
l-
t. .. IIl 1 J 1 1 J l mmm
-
Control
Congo
Males
Red
-
Control
Congo
Breeding
Per
Red
0
Control
Congo
Red
0
Contrc
Pairs
FIG. 1. The effects of oral administration of Congo red at 1 g/kg/day to the dam on Days 8- 12 of gestation on the reproductive physiology of the offspring. Data are from Experiment 1. Breeding pairs were established at 35 days of age using a male and a female from the same treatment groups. Data were collected up to 300 days of age. ( “:” denotes p < 0.05 different from control). The data analysis for “Mean litter size per breeding pair” did not include the four litters that did not breed, but the data from these pairs were included for other measures shown in this figure.
of the experimental designs and general methods employed in the present study is shown in Table 1. In the first experiment, the dams (PO generation) were treated with CR at 1 g/kg/day in water on Days 8-12 (Day 0 is the day of mating) by gavage and allowed to litter (the Fl). The numbers of dams in each group are shown in Table 2. Dams that had not delivered their litters within 24 hr of the expected delivery day were classified as “late.” On Postnatal Day 3, 8 control dams were randomly selected and assigned 3 pups/sex, randomly selected from the population of control pups. Six CR-treated litters were similarly established. On Postnatal Day 35, 14 control and 16 CR Fl breeding pairs were established. Both members of each breeding pair were from the same treatment group. Birth of the F2 pups was monitored over a 9-month period, during which the date of birth of the F2 pups and number of pups per litter were determined shortly after birth. At this time the F2 were removed and euthanized. At 300 days of age the Fl males were killed with carbon dioxide and necropsied and the females were euthanized. In males, testes, seminal vesicle, liver, kidney, and body weights were measured. A second experiment was designed to determine: (1) if a female factor was responsible for the infertility seen in the Fl CR-treated breeding pairs in the first study, (2) if the marked reduction in testis weight following prenatal CR treatment produced infertility, and (3) if the gonadal effects were reversible or became more severe with age. In this experiment, as in the first, the PO dams were dosed orally with CR at 1 g/kg/day or the vehicle (water) on Days 8-12 of gestation (sample sizes are shown in Table 2). At weaning, Fl mice were randomly assigned to breeding
pairs or unisexual housing conditions. For the Fl breeding component, 20 control and 19 CR-treated males were placed in breeding cages with 2 90-day-old untreated females/male. Pregnant females were allowed to deliver, pups (F2) were counted, litters were removed, and the female was replaced. These conditions were maintained until the males were necropsied when they were 360 days old. In order to assessthe fertility of the female offspring, 20 control and 20 CR-treated females were each paired with an untreated 90-day-old male. The males were replaced twice, when the females were 90 and 190 days of age. Fl females were necropsied at 9 months of age. In the second experiment, unisexual groups were also established in order to examine age-related changes in gonadal effectsin male and female mice. These groups consisted of 3 males/cage or 5 females/cage, with sample sizes of 45 CR males, 45 control males, 45 control females, and 30 CR females. At 90, 170, and 320 days of age approximately a third of the animals from each group were necropsied. In Experiment 2, body, testis, seminal vesicle, liver, and kidney weights were measured, a small sample of fluid from the vas deferens was taken, and sperm concentration was determined. One side of the reproductive tract, including the testis and epididymis, was fixed in Bouin’s, rinsed with alcohol, embedded in parathn, stained with H and E, and evaluated microscopically (Experimental Pathology Lab, NC). Testes were also examined histologically using a projecting microscope and the numbers of normal and completely atrophic tubules per cross section of testis were counted. In females, body weight was recorded at necropsy and the ovaries were fixed, as above, and examined for histopathological lesions.
414
GRAY ET AL.
TABLE 3 Maternal and Pup Data for Experiment 3
Con No. of litters Dam’s weight gain from Days 8 to 12 of gestation (g) % postimplantation loss
12
No. pups/litter on Day 1 Average pup weight on Day 1 k) Pup weight gain on Days 1-6 (g) % mortality on Days l-6 Pup weight gain on Days 6-29 % mortality on Days 6-29
11.7
(z&
(1.2) 1.66 (0.05) 2.11 (0.13) 0.03 (0.01) 36.2 (0.8) 6.0 (0.02)
CR (0.5 mg/kg/day) 6 4.3 (0.5) 10.8 (5.4) 10.0 (1.1) 1.I3 (0.04) 2.35 (0.15) 0.00 (0.00) 34.8 (0.7) 6.0 (0.00)
Note. Dams were dosed with 0.5 g/kg/day on Days 8-12 ofgestation with the azo dye CR or the vehicle (0.2 ml of water) (Con). There were no significant alterations of any of the maternal or neonatal measures. ’ Values are means (standard error of the mean).
A third experiment was designed with three objectives in mind: (1) to determine if a lower dose of CR affectedthe testis of the male fetus (CR was administered at 0.5 mg/k&day in 0.2 ml of water on Days 8-12 of gestation); (2) to compare the sensitivity of other indices of testicular function to testis weight; and (3) to determine if litter effectswere present in the data set. Pups were kept with their natural mothers, litters were randomly reduced (not culled) to seven pups (six males and one female, whenever possible), and on Postnatal Days 1, 6, and 29 the pups were weighed. The pups were weaned on Day 30, the dams were killed, and the numbers of implants were counted. Males from the third study were necropsied at 46-47 and 187- 190 days of age, at which time the left side of the reproductive tract was used for histopathology and body, testis, seminal vesicle, and cauda epididymal weights were recorded. In addition, cauda epididymal sperm reserves and sonication-resistant testicular sperm head counts were measured (see Gray et al, 1989b. for detailed methods). The females were not studied in this experiment. The preweanling data in the three experiments were analyzed using PRDC GLM, a general linear models procedure available on the SAS (SAS Institute, Inc., 1985), with the litter as the unit of statistical analysis. In Experiments 1 and 2. the postweanling data were analyzed as a completely randomized design because pups were not reared with their natural mothers and, hence, litter effectsare pooled with the general error term. In the third experiment, the postweanling data were analyzed as a nested analysis of variance, because pups were reared with littermates. This analysis removes the litter effect from the overall error term and the error variance is partitioned into a between-litter error variance (based on the variability between litters) and a within-litter error term (based on variability between littermates within litters). The treatment effect is a between-litter variable. The data from these experiments were also analyzed using nonparametric analyses (Fisher’s exact test). This was done because a comparison of the distributions of the testis weight data of control and CR males indicated that CR treatment increased
the variance and skewed the distribution of the data. In such situations, nonparametric analyses may provide a more accurate description of the data and be more sensitive. In this analysis, individual testis weights were compared against the mean value of the concurrent control group and those organs which weighed less than the control means minus three standard deviations were classified as abnormal. We have found that a testis that falls into this weight category typically contains atrophic tubules. Subsequently,
TABLE 4 The Effects of Prenatal Dye (Congo Red) Administration (1 g/kg/day, Days 8-12 of Gestation, by Gavage) on the Organ Weights, Seminiferous Tubular Atrophy, and Sperm Concentration in the Vas Deferens of Male Mice at Days 90, 170, 320, and 360 (Breeding Pairs) for Experiments 2a and 2b Days of age 90
170
320
360
Pooled
18 21
19 14
62 59
40.5 40.5
43.0 43.8
No. of males Control Congo red
12 12
13 12
Body weight (g) Control Congo red
39.9 40.7
43.5 44.3
46.9 41.3
Pooled SD, 3.67; treatment effect, NS; age, p < 0.0001; interaction, NS Testis weight (mg) Control Congo red
137 88
142 94
140 96
143 95
141 94
Pooled SD, 36.5; treatment effect, p < 0.0001; age, NS; interaction, NS No. of atrophic tubules/testicular cross section Control Congo red
1.1 13.2
0.8 21.2
0.8 18.2
3.6 14.1
1.7 16.8
Pooled SD, 13.1; treatment effect, p < 0.000 1; age, NS; interaction, NS No. of tubules/testicular cross section Control Congo red
316 227
270 217
274 227
283 226
284 225
Pooled SD, 52.4; treatment effect, p < 0.0001; age, NS; interaction, NS Vas deferens sperm concentration ( IO6 sperm/mg fluid) Control Congo red
1.84 1.38
1.70 1.18
1.76 1.14
1.24 1.04
1.60 1.17
Pooled SD, 0.68; treatment effect, p i 0.0008; age, 0.053; interaction, NS Seminal vesicle weight (mg) Control Congo red
415 389
524 527
754 642
587 551
587 547
Pooled SD, 132; treatment effect, NS; age, p c 0.000 1; interaction, NS Nore. SD, standard deviation; NS, not significant.
EFFECTS OF FETAL
EXPOSURE
TO CONGO RED
E‘IG. 2. Prenatal maternal administration of the azo dye Congo red (1 mg/kg/day, Days 8-12 of gestation) produces mild to severe testicular age due to hypospermatogenesis in affected seminiferous tubules in male mice. A normal testis is shown at the top, while the testis of a mildly affected CRbsed mouse is shown at the bottom (X 160).
GRAY ET AL.
416
the numbers of normal and abnormal testes in each treatment group were compared against the concurrent control using a Fisher’s exact test.
TABLE 5
Summary of the Nonparametrlc Analyses of the Data on Testis Weight and the Number of Tubules per Testicular Cross Section without Spermatogenic Cells
Maternal
Congo red
Control
Classification based on the number of atrophic tubules Experiment 2 No. of normal males No. of abnormal males Experiment 3 No. of normal males No. of abnormal males
RESULTS
60
1
39 20
31 0
9 9
p = 1.25
CR treatment did not adversely affect the viability or weight of the dams or their pups in any of the three experiments (Tables 2 and 3). Puberty (vaginal opening) was not delayed in CR-treated female offspring (data not shown). X 10m6 Male Necropsy Data x 1O-5
p = 2.4
In male offspring from the first experiment, testes weights were reduced by prenatal CR treatment (Fig. l), but body, seminal vesicle, liver, and kidney weights did not differ from control values, and no gross malformations were noted (data not shown). Similar effects were noted in the second and third experiments. The necropsy data from males housed in unisexual groups and killed at 90, 170, and 320 days of age and from the breeding males, killed at 360 days of age, from the second experiment are shown in Table 4. Body weight and seminal vesicle (with fluid) weight varied with age, but were not affected by CR treatment, and there were no interactions between these main effects. In marked contrast to body and seminal vesicle weights, testis weights did not change with age and they were consistently reduced by CR treatment at all ages (pooled means are 14 1 mg for control vs 94 for CR males). An example of a mild form of the testicular lesion seen in the CR-exposed males is shown in Fig. 2. In this experiment, the severity of hypospermatogen-
Classification based on testicular weight Experiment 1 No. of normal males No. of abnormal males Experiment 2 No. of normal males No. of abnormal males Experiment 3 No. of normal males No. of abnormal males
14 0
5
10 p = 1.5 x lo-5
62
34 25
0 31
p = 1o-8
4
14 p = 10-E
0
Note. A plot of the frequency distribution of the controls from all of our studies indicated that 98.5% of these males had fewer than 20 atrophic tubules per cross section. For this reason, testes with more than 20 completely atrophic tubules were classified as abnormal for the current analyses. Testes weights that were three standard deviations below the concurrent control mean were classified as abnormal.
22
I
I
I
I
I
and Neonatal Parameters
I
I
I
I
I
I
I
I
I
20 -
I
18-
I I -
3 c 0 z g @ '= 3 5
‘Severe’
Tubular
Congo
Red: 29%
0-q i
:
I
-++
\\
I
I
I
I
I
Control Males Congo Red Males
-
1614-
Control: 99% Congo Red: 52%
12lot. 61 4-
0
10
20
30
40
50
60
70
60
90
100110
120130140150160170160
Right Testis Weight,
190200210
(mg)
FIG. 3. A comparison of the frequency distributions of the testis weight and histology data from CR-exposed and untreated male mice from Experiments 2A and 2B. Pregnant females were dosed with the vehicle or the dye at 1 g/kg/day on Days S- 12 of gestation and their male offspring were necropsied at 90, 170, 320, or 360 days of age. The control and treated values were pooled across age because testis weights and the degree of hypospermatogenesis did not change from 90 to 360 days.
EFFECTS OF FETAL
EXPOSURE
TABLE 6 Male Offspring Necropsy Data for Experiment 3
Total No. of litters No. animals on Day 46 No. animals on Day 187 Body Weight (g) Day 46 Day 187 Pooled Testis weight (mg) Day 46 Day 187 Pooled SV weight (mg) Day 46 Day 187 Pooled Cauda epididymal weight (mg) Day 46 Day 187 Pooled Cauda epididymal sperm count (X106) Day 46 Day 187 Pooled No. of tubules per cross section of the testis Day 46 Day 187 Pooled No. of atrophic tubules per cross section Day 46 Day 187 Pooled Testicular sperm head count (X 106) Day 187
Con
CR
8 16 15
6 12 6
30.8 51.3 41.1
31.9 47.1 39.5
112 145 128
64 62 63
113 421 267
126 412 269
10.1 18.5 14.3 1.15 7.11 4.03 258 252 255 0.4 0.0 0.2
8.9 18.5 13.7 0.53 5.63 2.44 189 149 168 32.6 23.0 27.8
SDIEp
9.5/1.8, NS
33/42.1,
