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Morphological and biochemical changes in the adult male rat reproductive system following long-term treatment with 1 2-dibromo-3-chloropropane.

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THE ANATOMICAL RECORD 222:340-349 (1988)
Morphological and Biochemical Changes in the
AduIt Male Rat Reprod uctive System
Following Long-Term Treatment With
1,2-Dibromo-3Chloropropane
N. AHMAD, J.R. WISNER JR.,AND D.W. WARREN
Department ofAnatomy and Cell Biology (N.A.) and Department of Physiology and
Biophysics (J.R. W., D. W W),School of Medicine, University of Southern California,
Los Angeles, California 90033
ABSTRACT
Adult Long-Evans male rats were treated with various dosages of
pure or technical grade 1,2-dibromo-3-chloropropane
(DBCP), epichlorohydrin (Epi),
or allyl chloride (AC)for 1 , 3 , or 6 months on a daily basis. AC, which is the substrate
for the production of DBCP, and Epi, which is a contaminant andor metabolite of
DBCP, had no effect on any of the parameters of the male reproductive system
studied. The deleterious effects on male reproduction are therefore attributable
specifically to DBCP. The effects of DBCP were dose and duration dependent. At the
lowest dose (1 mgkg) DBCP did not have any discernible effects on the male
reproductive system. By 3 months of treatment at the intermediate dose of 5 mgkg,
the morphology of the testis ranged from normally appearing seminiferous tubules
to ones which contained Sertoli cells only. At 6 months of treatment there was a
reduction in the weights of the testes and sexual accessory glands. At the highest
dose, the majority of the rats showed advanced testicular regression by 1 month of
treatment. The most extreme testicular regression was observed in the 6-month
treatment group. Almost all of the seminiferous tubules of all of the rats were
composed of Sertoli cells only. In some of the animals, a few isolated seminiferous
tubules contained a n occasional spermatogonium or primary spermatocyte. Some of
the Leydig cells of the rats in this group showed morphological evidence of atrophy
as evidenced by the clumping of chromatin and paucity of stainable cytoplasm. This
was confirmed by lower levels of intratesticular testosterone, a significant reduction
in the number of luteinizing hormone (LH) receptors and increased serum levels of
LH and follicle-stimulating hormone (FSH). From these results we conclude that
DBCP is a specific male gonadotoxin and that the effects are not a result of contamination or metabolism. The effects appear to be a direct action at the testicular level
because feedback inhibition to the pituitary gland was adversely affected.
The compound 1,2-dibromo-3-chloropropane (DBCP) is
a halogenated aliphatic hydrocarbon which has been
quite extensively utilized as a n effective nematocide
until it was discovered that it had profoundly harmful
effects on the male reproductive system. While its use
has been banned in the United States, the study of this
chemical is of interest because it has specific effects on
the testes and thus, can be used as a probe for the
investigation of male reproductive function.
DBCP is produced by bromination of allyl chloride
(AC) and a t times dehydrated by the use of epichlorohydrin (Epi), which is also a metabolite of DBCP. Epichlorohydrin, in turn, is metabolized to alpha-chlorohydrin
(ACH). Therefore, the production or technical grade of
DBCP is invariably contaminated with unknown quantities of AC and sometimes Epi. The effects of AC on
male reproductive morphology are essentially unknown.
Epi and its metabolite ACH on the other hand, are
known to cause infertility in male animals of various
0 1988 ALAN R. LISS, INC.
species (Erricson and Baker, 1970; Cooper et al., 1974;
Jones et al., 1969; Edwards and Jackson, 1970; BrownWoodman et al., 1979).
DBCP is structurally similar to ACH and produces
essentially the same urinary metabolites as ACH (Jones
et al., 1979; Kato et al., 1979). Therefore, when technical-grade DBCP, with its inherent contaminants, is used
it is difficult to make a determination a s to which substance or metabolite was responsible for the deleterious
effects.
DBCP has been shown to cause testicular regression
in men working in the manufacturing plants (Whorton
Received February 15, 1988; accepted May 24, 1988.
Address reprint requests to Nazir Ahmad, Ph.D., Department of
Anatomy and Cell Biology, School of Medicine, University of Southern California, 1333 San Pablo Street, Los Angeles, CA 90033.
This study was supported by funds from Amvac Chemical Corporation, Los Angeles, California.
LONG-TERM EFFECTS OF DBCP IN THE MALE
et al., 1977; Biava et al., 1978; Lantz et al., 1981; Potashnik et al., 1979; Lipshultz et al., 1980; Cortes-Gallegos
et al., 1980).The exposure in these workers had occurred
for the most part via inhalation, skin contact, and possibly by ingestion in the drinking water. While there is
some correlation with the duration of exposure and testicular atrophy among the workers exposed to DBCP, it
is not possible to make a determination of the exact
amount of exposure.
In order to duplicate the mode of exposure by human
subjects, experimental studies in animals were designed
to administer DBCP via inhalation, drinking water or
diet. The effects of DBCP have been studied by administration via food in rats (Torkelson et al., 1961),inhalation in rats (Torkelson et al., 1961; Segusa et al., 1982;
Rao et al., 19831, and rabbits (Rao et al., 19821, in water
in rats (Torkelson et al., 1961; Faydish et al., 1970;
Rahmatullaev, 1971), in rabbits (Foote et al., 1986a,b),
and by gavage in rats (Amann and Berndtson, 1986).
DBCP has also been studied by subcutaneous injections
in rats (Kluwe et al., 1983; Warren et al., 1984a).
The addition of technical grade DBCP to the diet at 5
to 1,350 ppm for 90 days had no effect on testes in the
rats, (Torkelson et al., 1961). Delivery of DBCP a t 70
m g k g per day in water for 45 days caused necrosis of
testes, (Faydish et al., 1970). The same observation for
the preceding experimental protocol was reported by
Rakhmatullaev (1971). In the later study, five rats died
during the course of the study and the remaining five
rats showed testicular atrophy at the conclusion of the
experiment. However, a dose of 5 mgkglday in water for
8 months resulted in a n increased weight and length of
the testes. On the other hand, Foote et al. (1986a,b),
noted decrease in sperm count of rabbits that were given
15 m g k g of DBCPlday15 dayslweek for 70 days in water.
They noted a n increase in the level of serum folliclestimulating hormone (FSH) a t the highest dose, but
there was no effect on the values of luteinizing hormone
(LH), testosterone, and fertility index. Histologically
these investigators noted reduction in the number of
germinal cells associated with each Sertoli cell a t a
minimal dose of 1.88 mgkg.
In a recent study Amann and Berndtson (1986)administered 0 to 15 mglkg of DBCP per day for 77 days to
rats by gavage. At the highest dose level they found no
effect on the serum levels of FSH and testosterone. In
this study, sperm motility and ability to accomplish
fertile mating was not affected. On the other hand, when
DBCP was administered to rats at a concentration of 10
ppm continuously for 10 days by inhalation, necropsy a t
various intervals following the treatment showed completely regressed testes (Segusa et al., 1982). When the
rats were exposed to 40 ppm of DBCP by inhalation for
7 hourslday, 5 dayslweek for a total of 50 exposures, the
majority of the rats died by the 4th week of treatment
(Torkelson et al., 1961). The testes of these rats were
found to be quite atrophic. In the same report, exposure
a t 12 ppm for 7 hoursldayl5 dayslweek for 50 to 66
exposures, the authors noted regression of spermatogenesis, a n increase in the number of Sertoli cells, abnormal
germinal cells, and a concomitant decrease in the number of mature germinal cells. However, the animals were
not rendered azospermic. In a similar study, Rao et al.
(1983) administered DBCP by inhalation at concentrations of 0-10 p p d 6 hoursldayl5 dayslweek for 14 weeks
341
and noted only moderate testicular atrophy which did
not effect the ability of the treated rats to impregnate
females. When these authors used the same protocol in
rabbits, Rao et al. (1982), they noted atrophy of testes,
epididymides, prostate glands and external genitalia. At
the conclusion of 8 weeks of treatment at the highest
dose studied, the testes showed severe regression and
were comprised of predominantly Sertoli cells. Only few
tubules showed some germinal elements in addition to
the Sertoli cells. After a period of recovery some tubules
showed normal spermatogenesis while others were devoid of germinal elements.
In most of the above-mentioned studies technical-grade
DBCP was utilized and the nature and concentration of
impurities was not monitored. Furthermore, in many of
the experiments, the exact amount of DBCP delivered
to the animals is difficult to evaluate.
The purpose of this study was to elucidate the development of the effects of DBCP, as differentiated from
the contaminants or metabolites, on the morphology and
biochemistry of the adult male reproductive system, as
measured at varying times during a 6-month treatment
period.
MATERIALS AND METHODS
Animals
Adult male Long-Evans rats, 2 months of age, were
obtained from Simonson Laboratories (Gilroy, CA). The
animals were housed five per cage in a room with 12
hours of light and 12 hours of darkness. The rats were
given Purina Lab Chow and tapwater ad libitum. All
animals were weighed and ear-punched for identification prior to the initiation of injection protocol.
Chemicals
Pure and technical-grade DBCP as well as Epi and AC
were obtained from the Amvac Chemical Corporation
(Los Angeles, CAI. Assessment of the purity of the chemicals was performed by GHT Laboratories (Brawley, CA).
The pure DBCP contained 0.4% AC, and the technicalgrade preparation was contaminated with 3.2% AC.
Since DBCP is a hazardous substance, extensive safety
procedures were utilized throughout the study as reported earlier (Warren et al., 1984a).
Rat luteinizing hormone (LH) and follicle-stimulating
hormone (FSH) radioimmunoassay (RIA) kits were gifts
from the National Hormone and Pituitary Program of
the University of Maryland, School of Medicine, and the
National Institute of Diabetes, Digestive and Kidney
Diseases. Burro antirabbit gamma globulin was purchased from Immunocorp (Sunnymead, CA).
All test chemicals were dissolved in corn oil (Sigma
Chemical Company, St. Louis, MO). Fresh preparations
were made on a weekly basis. Since breakdown of DBCP
was a potential problem, the stock solution of DBCP as
well as various dilutions were periodically analyzed by
gas chromatography for accuracy of dilutions and possible changes. No breakdown of the stock solution or dilutions were encountered during the course of these
studies.
Experimental Design
The experimental design is depicted in Table 1. While
initial studies were performed using pure and technical
grades of DBCP, as was shown in our earlier study
342
N. AHMAD ET AL.
TABLE 1. Experimental design'
No. of animals
Duration of
treatment
(months)
1
3
6
DBCP
(mgkd
1 5 25
10
10
30
10 8
10
6
30 26
Epi:
25mgkg
AC :
50mgkg
Control
5
5
1.5
5
5
-
13
17
to 1-and 5-mgkg-DBCP-treated rats as well as controls
treated with Epi or AC (Fig. lg). By three months of
treatment, body weights of 5- and 25-mgkg-DBCPtreated groups were significantly (P<0.05) lower than
the those treated with 1 m g k g DBCP, Epi, or AC
(Fig. lh). The relationships between the body and organ
weights for the 6-month treatment regimen were similar to the 3-month experiment, as has been reported
earlier (Warren et al., 1984a).
'The No. of DBCP treated animals represents the combined total of
rats treated with both pure and technical-grade reagent.
(Warren et al., 1984a1, and confirmed in this study, no
differences in effects between the two grades of DBCP
were found for any parameter. Additionally, since no
differences were found in any of the parameters studied
between control and AC and Epi treatments, these compounds served as control groups at 1 and 3 months'
treatment duration. All of the animals were weighed a t
the onset of the experiment and thereafter on a weekly
basis. The dosage of the test substances was calculated
on the mean body weight of each group at weekly intervals. All animals were injected subcutaneously in the
back of the neck in volumes ranging from 20 to 100 pl.
For accuracy and speed of injections, Hamilton microliter syringes fitted with automatic dispensing units were
utilized.
At the conclusion of the experiments, the rats were
weighed, killed by decapitation, and trunk blood collected for subsequent hormonal determinations. After
the blood clotted, serum was separated and stored a t
-20°C for LH and FSH determinations. Paired testes,
prostate glands and seminal vesicles were weighed. One
testis was snap frozen and stored a t -20" C for intratesticular LH receptors and testosterone determinations.
The other testis, prostate glands, and seminal vesicles
were fixed in Bouin's fluid and prepared for histological
study as described earlier (Ahmad et al., 1973). Briefly
the tissues were embedded in nitrocellulose, sectioned
a t 7 pm on a sliding microtome, mounted, and stained
with periodic acid, Schiff s (PAS) and hematoxylin.
Hormones and Receptors
Serum concentrations of LH and FSH were measured
by RIA as previously described (Warren et al., 1984a).
The sensitivities of the assays were 4.0 ng LWml and
60 ng FSH/ml. All samples were analyzed in a single
run for each hormone. The interassay variation were
< 10% for each of the hormone measured. LH receptors
and intratesticular testosterone were measured as previously described (Warren et al., 1984b). The data was
analyzed by utilizing Duncan's new multiple range test,
Steel and Torrie, (1960). The significance of differences
was calculated a t a level of P< 0.05.
RESULTS
Body Weights
There were no significant differences between the pure
and technical-grade of DBCP in terms of body or organ
weights as well as all of the other parameters tested at
any duration studied. Therefore, the results for these
two preparations are presented collectively. The results
of the body weights are shown in Figure 1.At the end of
1month the rats treated with 25 m g k g of DBCP showed
significantly (P< 0.05) lower body weights as compared
Testicular Weights
The testicular weights of rats treated with the highest
dose of DBCP for 1 month were significantly (P<O.O5)
lower than those of all other groups (Fig. la). By 3
months of treatment, the testes of rats treated with 5 or
25 m g k g were significantly lower (P<O.O5) than all
other groups, with the latter showing the greatest loss
(Fig. lb). The testicular weights of the 1-mg-treatment
group were not significantly different than those of the
control groups treated with Epi or AC a t any time period
(Figs. la,b). At the conclusion of 6 months of treatment,
the relationships between testicular weights of the varying doses of DBCP treated rats were similar to those
obtained after 3 months; however, the decrease in testicular weights was more pronounced at the highest dose
a s reported earlier (Warren et al., 1984a).
Prostate Glands and Seminal Vesicles
At 1- and 3-month intervals, the prostate glands of
rats treated with the highest dose of DBCP were significantly lower (P<O.O5) than those of all other groups
(Figs. le,f). Rats treated with the lower dosages showed
no weight loss of prostate glands as compared to those
treated with Epi or AC. The same was the case with the
weights of the seminal vesicles (Figs. lc,d).
Histology
Normal controls
The histological appearance of all of the reproductive
organs in control rats a t the end of 6 months was normal. The testes were composed of large seminiferous
tubules displaying various stages of spermatogenesis.
The interstitial tissue was composed of nests of Leydig
cells with vesicular nuclei and stainable cytoplasm. The
prostate glands and seminal vesicles were characterized
by large secretory units filled with secretory product
and lined by tall columnar epithelial cells. In the case of
the prostate glands, the epithelial cells showed a prominent supranuclear negative image of the Golgi apparatus, which is characteristic of normally functioning
gland. Epididymis was lined by tall columnar epithelium, and its lumen was filled with mature spermatozoa.
The histological appearance of all of the reproductive
organs of rats treated with 25 m g k g of Epi or 50 m g k g
of AC for the three durations studied was indistinguishable from that of normal controls a t 6 months (Figs. 2,
4,6).
1 mglkg DBCP
The histological appearance of all of the reproductive
organs from rats treated with 1 m g k g DBCP for all of
the durations studied was indistinguishable from that
of normal controls, with the exception of one rat that
showed a few regressing seminiferous tubules a t the end
of 6 months of treatment.
343
LONG-TERM EFFECTS OF DBCP IN THE MALE
,-.
or
n
f
v
c
r
CJI
._
0.6
a,
c
0
c
in
P
LL
T
+
c
One Month Treatment
Fig. 1. The effects of various dosages of DBCP, 25 m g k g Epi, and 50
m g k g AC on reproductive organ and body weights of rats treated for
1
w
lDEP
25DBcP
c
Ep(
Three Month Treatment
1 (a, c, e, g) or 3 (b, d, f, h) months.
and AC groups (P<0.05).
* Significantly different from Epi
5 mglkg DBCP
rats, a small number of the tubules showed normal
In animals treated for 1 month with 5 m g k g DBCP, stages of germinal development, while the majority of
the histological appearance of all of the reproductive the tubules had undergone advanced regression. As far
organs were indistinguishable from that of controls, with as the “normal”-appearing seminiferous tubules in the
the exception of one rat that showed a few regressing DBCP-treated rats are concerned, we did not count each
tubules. The histological appearance of testes of four cell type; therefore, it is quite likely that the relative
rats treated with 5 m g k g of DBCP for 3 months was number of various spermatogenic cell types may not be
comparable to that of normal controls. In four other rats, comparable to normal controls. The morphological apa vast majority of the seminiferous tubules showed nor- pearance of the sexual accessory glands of rats with
mal advanced stages of spermatogenesis. However, a normal or regressing testes was similar to that of the
number of tubules showed varying degrees of regres- untreated animals (Figs. 5, 7). The epididymidis of rats
sion. This ranged from incomplete cellular association with regressing testes contained varying amounts of
to tubules comprised of Sertoli cells only (Fig. 3). In two immature germinal cells that had been prematurely
345
LONG-TERM EFFECTS OF DBCP IN THE MALE
sloughed off. The epididymides of all other rats were
like those of control animals.
The results of histological study of the testes of rats
treated with 5 m g k g of DBCP for 6 months were similar
to those reported after 3-months. All of the reproductive
organs of one-half the group were like those of normal
controls. The majority of the seminiferous tubules of six
rats showed normal advanced stages of spermatogenesis, while only few isolated tubules showed varying degrees of regression. In five other rats the majority of the
tubules showed advanced regression, with only few tubules showing normal stages of spermatogenesis. Aside
from the fact that the luminal contents of the epididymides of rats with regressing tubules showed a paucity
of mature spermatozoa and a n increased number of immature germinal cells, all other reproductive organs
were morphologically comparable to those of normal
controls.
25 mg/kg DBCP
The testes of four of the rats treated with 25 m g k g for
1 month were histologically like those of normal rats.
The rest of the rats showed varying degrees of advanced
testicular degeneration with some showing seminiferous
tubules that had regressed to Sertoli cells only. While
the weights of the sexual accessory glands were decreased as compared to normal animals, histologically
the secretory units appeared well maintained.
By 3 months of treatment with 25 m g k g of DBCP, the
seminiferous tubules of all of the rats were virtually
devoid of all germinal elements and had regressed to
Sertoli cells only. While morphologically nests of Leydig
cells were identifiable, some clumping of nuclear chromatin, indicative of atrophy, was noted. The sexual accessory structures were like those described for the 1month-treated rats.
At the conclusion of 6 months of treatment with 25
m g k g of DBCP, only one rat had a few tubules with
some advanced-stage spermatids. The testes of the remaining rats in this group were characterized by smalldiameter seminiferous tubules which were lined by Sertoli cells only (Fig. 8). In a few isolated tubules of some
of the rats, it was possible to locate a n occasional spermatogonium or primary spermatocyte, but for the most
part spermatogenic cells were not present (Fig. 10). The
Leydig cells were small and showed clumping of nuclear
chromatin (Fig. 9). In animals with regressed seminiferous tubules, the lymphatic spaces had increased signifiFig. 2. Seminiferous tubules showing normal maintenance of spermatogenesis from a rat that was treated daily for 6 months with 25
mgkg of Epi. ~ 7 5 .
Fig. 3.Seminiferous tubules with some showing advanced regression
and others showing normal spermatogenesis from a rat that was treated
daily for 6 months with 5 m g k g of DBCP. ~ 7 5 .
Fig. 4. A well-maintained secretory unit of a prostate gland of a rat
treated with Epi as in Figure 2. x 190.
Fig. 5. Several normally maintained secretory units from a prostate
gland from a rat treated with 5 mgkg of DBCP as in Figure 3. ~ 7 5 .
Fig. 6. A small segment of a normal-appearing seminal vesicles of a
rat treated with Epi as in Figures 2 and 4. x 75.
Fig. 7. Morphologically normal-appearing seminal vesicles of a rat
treated with DBCP as in Figures 3 and 5. ~ 7 5 .
cantly (Figs. 3, 8, 9, 10). The prostate glands were
composed of acini of variable sizes. For the most part
the secretory units appeared to be smaller than those of
normal controls. Although most of the acini were lined
by low cuboidal epithelium, their lumen was filled with
PAS-positive secretory product as is the case in normal
glands (Fig. 11).The seminal vesicles appeared to be
smaller but morphologically maintained with characteristic secretion in their lumen (Fig. 12). The epididymal,
epithelium showed morphological maintenance; however, their lumen was generally filled with homogeneous or clumped mass of PAS positive material. There
was no evidence of mature germinal elements, as is the
case in normal rats.
Hormones and Receptors
Figure 13 shows the results of serum LH and FSH
after 1and 3 months of treatment with varying doses of
DBCP as compared to AC and Epi controls. While LH
levels a t all doses of DBCP were not significantly different than controls after one month of treatment, all doses
of DBCP were associated with significantly elevated
serum levels of LH when compared to controls after 3
months (Fig. 13c,d). On the other hand, the FSH titers
of the group treated with 25 m g k g were significantly
higher (P<O.O5) than all other groups a t both 1 and 3
months (Figs. 13a,b).
Figure 14 depicts the LH receptor and intratesticular
testosterone levels in testes of animals treated for 1, 3,
and 6 months with 25 m g k g of DBCP as compared with
Epi and AC controls. Only the 25-mgkg dose of DBCP
evoked a consistent effect and thus these studies were
limited to this dose level. Both parameters were significantly decreased (P<0.05) by greater than 50% at 3 and
6 months of treatment when compared to Epi and AC
controls. No significant differences were noted after only
1month of treatment.
DISCUSSION
The results of this study clearly show that DBCP is
very specifically gonadotoxic in the male. The effects are
dose and duration dependent. A dose of 25 m g k g given
daily caused testicular regression in a significant number of animals by 1and 3 months’ duration. By 6 months
of continuous administration, most of the testes had
regressed to Sertoli cells only. In this entire group, only
a few seminiferous tubules contained any germinal cells.
At 5 mgkg, gonadotoxic effects were apparent a t a progressively greater extent, depending on the duration of
administration. The lowest dose of 1m g k g had no deleterious effects at all at the durations studied. The fact
that there was no significant difference between the
technical grade of DBCP and the highly purified preparation of DBCP and that Epi and AC had no effects
indicates that the primary effects are attributable to
DBCP and not the possible contaminants or metabolites.
It is of interest to note that AC is a substrate of DBCP
and has not been previously studied for its effects on
reproduction. While DBCP has specific and profound
effects on male reproduction, AC does not possess the
same deleterious properties.
It is very difficult to correlate the results of our study
with other reports in the literature because of profound
differences in the experimental protocols, use of different experimental animals, dosages of DBCP used, and
347
LONG-TERM EFFECTS OF DBCP IN THE MALE
Ib
I
*
*
--
Fig. 13. Serum concentration of FSH at 1 month (a), 3 months (b);
LH at 1 month (c), 3 months, (d) of rats treated with various dosages
routes of administration. The results of various studies
range from total testicular regression to no effect on
reproduction, as has been previously described.
In terms of primary gonadotoxic effects of DBCP, our
results are in agreement with the results reported by
Faydish et al. (1970), which showed total testicular
regression when 70 m g k g of DBCP was administered
via water intake for 45 days. Similarly, inhalation of
DBCP at 10 ppm continuously for 10 days caused the
seminiferous tubules to regress to Sertoli cells only (Segusa et al., 1982).
The mode of the administration of DBCP and the
reliability of the amount delivered appears to be of considerable importance in terms of gonadotoxic effects observed. Torkelson et al. (1961), found no effects when
1,350 ppm of DBCP was given in food to rats for 90 days.
Similarly, 5 m g k g given in water to rats for 8 months
(Rakhmatullaev, 1971)and 15 m g k g for 77 days (Amann
100,
I
a
0AC
EJ €pi
DBCP
80 -
1.000
,
b
Fig. 8. Testis of a rat treated daily with 25 m g k g of DBCP for 6
months. The entire testes were composed of seminiferous tubules which
had regressed to Sertoli cells only. ~ 7 5 .
Fig. 9. A higher magnification of a testis of a rat treated with DBCP
as in Figure 8. A portion of the seminiferous tubule (ST) shows a
thickened wall and Sertoli cells. The arrow points to a cluster of
atrophic Leydig cells which show clumped chromatin and sparse cytoplasm. Arrowhead points to a macrophage. ~ 3 0 0 .
0500
-
Fig. 10. Another view of a testis treated with DBCP as in Figures 8
and 9. A seminiferous tubule (ST)with mostly Sertoli cells and a
solitary spermatogonium (arrowhead) is shown. ~ 3 0 0 .
0 000
Fig. 11. Secretory units of a prostate gland treated with DBCP as in
Figure 10. The acini are lined by columnar epithelial cells with supranuclear halo which is characteristic of active secretory cells of a normal
prostate gland. x 190.
Fig. 12. Morphologically normal-appearing seminal vesicles of a rat
treated with DBCP as in Figures 8-10. x 75.
1
3
Months of Treatment
Fig. 14. LH receptor contents (b) and intratesticular testosterone
levels (a) for rats treated with DBCP 25 mgkg, Epi 25 mgikg, Ac 50
mgikg for 1,3, or 6 months. * Significantly different from other groups
(P<O.Ol).
348
N. AHMAD ET AL.
animals died and their testes showed advanced regression. However, in the same study, when the rats were
exposed to 12 ppm of DBCP for a total of 50-60 exposures, the testicular regression was moderate and the
rats were not azospermic a t the conclusion of the
experiment,
In terms of delivery via food or water, for the most
part, the animals exposed to higher concentrations of
DBCP reduced their consumption significantly. In studies by Foote et al. (1986a,b),this problem was eliminated
by having the rabbits consume all of the water containing DBCP within a n hour each day. However, testicular
regression in this study was quite moderate and none of
the animals were azospermic.
While exposure of human workers by DBCP occurred
by inhalation and/or skin contact and possibly by ingestion in water that resulted in various degrees of testicular regression (Whorton et al., 1977), experimental
verification of the primary effects of DBCP are best
studied by administering precise amounts of the test
substance a t regular intervals. In the present study, the
design of the experiment was to achieve delivery of a n
accurate amount of DBCP at regular intervals for the
various durations studied. This was accomplished by
injecting various dilutions of DBCP with the aid of syringes equipped with automatic dispensing mechanisms.
One aim of this study was to ascertain possible effects
of the contaminants of DBCP on male reproduction. To
that effect, Epi at a dose of 25 m g k g or AC at a dose of
50 m g k g was administered daily for 1, 3, or 6 months
along with pure and technical grades of DBCP. Epi as
well as AC showed no effects at the testicular level a t
all durations studied. Additionally, there were no effects
on the body weights, prostate glands, and seminal vesicles weights as compared to untreated controls. The
hormonal values of LH and FSH also did not differ from
normal controls.
Epichlorohydrin is a known male reproductive toxin
and causes infertility by its effects at the level of epididymis (Erricson and Baker, 1970). In addition, it has been
shown that Epi is hydrolyzed to ACH in vivo (Jones et
al., 19691, which renders the epididymal spermatozoa
infertile by interfering with their ability to metabolize
glucose (Brown-Woodman et al., 1979). It has also been
indicated that Epi and ACH cause infertility in males
by a similar mechanism (Jones et al., 1969; Cooper et
al., 1974). Additionally, some investigators claim that
these compounds, when given a t high dosages and over
long periods of time can cause testicular regression as
well. We have not monitored the epididymal effects of
Epi or AC. However, we did not notice any testicular
changes in animals treated with these two substances.
In a study by Kluwe et al. (1983), adult rats were
injected with a single sublethal dose of DBCP (80 mgl
kg), ACH (125 mgkg), or Epi (75 mgkg); and Severe
testicular atrophy was noted 75 days following the treatment with all three test substances. In this study, epididymal granulomas and spermatocoeles were formed by
ACH and Epi and not DBCP. The results of our experiments with DBCP, in terms of its effects on testes and
epididymides, are in agreement with this report. However, in terms of effects of Epi on testes, in our experiments 25 mgkglday for 180 days, for a total of 4,500
mgkg, had no effect on testes. The difference in results
may be explained in part by the differences in the exper-
imental protocol and the use of different strains of animals in these two studies. Furthermore, it is possible
that the administration of small doses of Epi over the
period of 24 hours allowed our animals to metabolize
Epi by the time next dose was administered.
The authors in the above-mentioned study concluded
that the metabolism of DBCP to ACH and Epi in not
sufficient to explain the gonadotoxic effects of DBCP.
We agree with this conclusion based on our results. In
their study all 3 test substances caused testicular regression, while in our study only high dosages of DBCP
caused testicular atrophy. In both studies DBCP did not
cause any morphological damage to the epididymis.
Thus, it can be concluded that Epi and/or ACH are
directly responsible for the lesions encountered in the
epididymis. The fact that in our study only DBCP caused
testicular regression shows that DBCP and not its possible contaminants or metabolites are responsible for its
primary effects on male gonads.
In the DBCP-treated animals which showed maximal
testicular regression, the seminiferous tubules were
composed of Sertoli cells only. The loss of germinal elements results in a dramatic decrease in the size of seminiferous tubules. Invariably, this regression results in
shrinkage in length as well a s width of the seminiferous
tubules. Additionally, since the germinal elements are
not present and therefore do not occupy the Sertoli cell
cytoplasm, the number of nuclei of Sertoli cells per cross
section of a seminiferous tubule, in this shrunken state,
is invariably greater. Some investigators have interpreted this to mean that testicular regression results in
a n increased number of Sertoli cells (Torkelson et al.,
1961). We have also noted this apparent “increase” in
the number of Sertoli cells nuclei per cross section of a
seminiferous tubule. However, it is a well known fact
that the number of Sertoli cells does not increase after
the animal reaches two weeks of age (Clermont and
Perey, 1957; Steinberger and Steinberger, 1971).
The fact that the Sertoli cells appear to be increased
in number and morphologically intact does not mean
that they are functionally viable. Increased levels of
FSH indicate a loss of feedback inhibition from products
of the seminiferous tubules and specifically the Sertoli
cell (Steinberger and Steinberger, 1976). In addition, the
loss of germinal cells might well reflect damage to the
Sertoli cell, rendering it unable to support the developing germinal cells.
The same shrinkage phenomenon results in the aggregation of larger nests of Leydig cells that are visualized
in the interstitium of regressed testes. However, there
is no evidence for an increase in the number of Leydig
cells as a result of mitotic proliferation of Leydig cells.
In our study, in spite of the fact that the LH values of
the DBCP-treated rats were higher than the control
groups, we did not notice any Leydig cell hypertrophy
or hyperplasia. As a matter of fact, some of the Leydig
cells of DBCP-treated rats appeared atrophic, as evidenced by clumping of their nuclear chromatin (Fig. 9).
The level of testosterone secreted by these Leydig cells
must have been minimal because the seminal vesicles
and prostate glands of the rats treated with 25 mglkg,
a t all intervals studied, were smaller than all other
groups. Additionally, LH levels were elevated after 3
months of treatment indicating decreased levels of testosterone. In the rats treated for 6 months with the high
LONG-TERM EFFECTS OF DBCP IN THE MALE
dose of DBCP, the testicular content of testosterone as
well as the LH receptors in the testes were significantly
reduced as compared to untreated rats. However, it
should be noted that there was evidence of androgen
secretion because these miniature sexual accessory
gIands of rats treated with the highest dose of DBCP for
6 months were lined by morphologically well maintained epithelial cells and there was secretory product
in the lumen of the glandular acini.
The results of this study show that DBCP is a specific
male gonadotoxin. The damaging effects of DBCP are
specific to this compound and not a result of contamination with AC, its substrate, or its metabolite, Epi. The
effects appear to be a t the level of the testis because
feedback inhibition of the anterior pituitary was compromised as evidenced by increased levels of LH and
FSH a t the highest treatment dose level. While there is
evidence to show that the Leydig cells and Sertoli cells
were affected, the design of these experiments does not
permit us to attribute the effects directly to specific cell
types.
349
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