Toxicology, 75 (1992) 39-50 Elsevier Scientific Publishers Ireland Ltd.
39
Effects of 2,5-hexanedione on the ovary and fertility. An experimental study in mice G. Siracusa a, A. B a s t o n e b, M. Sbraccia b, L. Settimi b, C. M a l l o z z i b, E. M o n a c o c a n d N. Frontali b aDepartment of Public Health, Section of Anatomy & Cell Biology, 2nd University of Rome, blstituto Superiore di Sanita ', Rome and CLaboratory of Animal Sciences, ENEA, CRE Casaccia, Rome (Italy)
(Received January 28th, 1992; accepted June 24th, 1992)
Summary Sixty-day-old virgin female Swiss CD1 mice were treated with 1.5% 2,5-hexanedione in their drinking water; control mice received tap water; duration of treatment was either 4 or 6 weeks. Under these conditions the treated mice did not show any clinical symptoms although electromyography revealed some signs of polyneuropathy. Protein and DNA content per mg of ovarian tissue in treated mice were not significantly different from controls. Histological examination of ovarian sections at the light microscope level showed no significant alterations after exposure. A morphometric study revealed a statistically significant reduction in the number of growing oocytes after 6 weeks of treatment. For fertility studies three groups of 15 female mice each were treated for 0, 4 or 6 weeks as above and then permanently housed with untreated proven breeder male mice (one male per female); cages were checked daily for newly born mice. All litters appeared normal by gross examination. During the first 14 weeks of continuous mating the mean litter size (number of newborns per litter) remained about 1i.4 in all groups; this number subsequently began to decrease. Control and 4-week treatment regression curves did not differ statistically, while the slope of the 6-week line was significantly steeper, indicating a faster decrease in litter size over time and a shortening of fertile life in the latter group of treated females.
Key words: 2,5-Hexanedione; n-Hexane; Ovary; Fertility; Reproductive toxicology
Introduction n-Hexane has several applications in industry as a solvent, especially in glues, varnishes, cements and inks and is also used to extract oils from various seeds. Persons exposed to the solvent in poorly ventilated shoe and leather workshops and factories in Japan, Italy, France and Spain have developed severe forms of polyneuropathy; the employees were mainly women [1,2]. This neurologic condition has been reproduced in experimental animals treated with pure n-hexane (rat) or with its neurotoxic metabolite 2,5-hexanedione (cat, rat and mouse) [3]. A largely accepted Correspondence to." G. Siracusa, Dipartimento di Sanita' Pubblica e Biologia Cellulare, II Universita' di Roma, Via O. Raimondo, 00173 Roma, Italy. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
40 hypothesis regarding the mode of action of this metabolite is that it binds to some cytoskeletal or cytoskeleton-associated proteins, altering their function (for a review see Ref. 4). Treatment of male rats with 2,5-hexanedione (2,5-HD) [5] or with n-hexane [6] has been found to cause testicular alterations in addition to polyneuropathy; practically no information is, however, available on the experimental effects of these compounds on the female reproductive system. The present work is a contribution towards filling this gap. Fewer quantitative biological markers have been elaborated for female than for male reproductive toxicity studies; histological assessment of ovarian follicle number is one endpoint in the female system [7], another is the total reproductive capacity test elaborated by Generoso et al. [8]. In most cases such work has been performed with mice rather than rats as experimental animals, mainly because of their better characterized reproductive parameters. In our study, the dose and the timing of treatment of female mice with 2,5-HD were chosen in such a way as to produce mild signs of polyneuropathy, as revealed through electromyography in a preliminary experiment. Materials and methods
Animals and treatments Virgin female Swiss CDI mice (Charles River, Italy) were randomly assigned to experimental and control groups and housed in plastic cages (6-10 per cage) at a room temperature of21°C ± 1 with a 12-h dark/light cycle (60% humidity); they had free access to food (a standard Vogt Miller diet in pellets) and water. Treatment began after a 10-day period of acclimation, when the mice were 60 days old, with the addition of 1.5% (v/v) 2,5-HD (Fluka AG, Buchs, Switzerland, re-purified by reflux distillation) to the animals' drinking water; duration of treatment was either 4 or 6 weeks. In each experiment all animals began treatment on the same day, with the exception of the fertility studies, where all ended on the same day (see below). The mice were weighed at the start of treatment and then at weekly intervals. Electrophysiological studies One experimental and one control group were employed, each consisting of 14 mice. The animals were subjected to electromyography at the onset and at weekly intervals for up to 5 weeks. Motor conduction velocity and distal latency time were measured on the tail nerves according to Miyoshi and Goto [9]. Biochemical studies For each time interval considered in the biochemical and morphological studies one experimental and one control group were used, each consisting of 6-10 mice. At given intervals, treated and control mice were sacrificed by cervical dislocation and the ovaries quickly removed. The right ovary was placed in a watchglass containing a physiological saline solution and was freed from surrounding adipose and connective tissue under a dissection microscope. It was then quickly blotted, weighed and homogenized using a glass Elvejhem Potter apparatus (Teflon pestle) in 10 ml of saline buffer (NaC1 2 M, Na2HPO4 0.05 M, EDTA 2 x 10-3 M, pH 7.4) per
41 g of tissue. The homogenate was assayed for protein [10] with BSA as a standard and for DNA [11]. Measurements were performed with a Zeiss PM7 spectrophotometer and with a Perkin Elmer LS-3 fluorescence spectrometer, respectively.
Morphological studies Light microscopic observation and oocyte morphometry were performed on left ovaries fixed in Bouin's medium, embedded in paraffin, serially sectioned at 6 #m and stained with haematoxylin and eosin. Oocytes were classified according to cellular diameter in three categories: small (resting oocytes, 70 #m) [12]. The small oocytes were counted in every twelfth section, the medium and large in every sixth; nucleoli were used as markers for all three categories of oocytes. The total number of oocytes per ovary was calculated according to Abercrombie [13]; all counts were made on one ovary and doubled to express the total number of oocytes per animal.
Fertility studies In order to study the effect on total reproductive capacity of a 4- or 6-week treatment with 2,5-HD, 8-week-old virgin female mice were randomly divided into three groups (each consisting of 15 mice). Animals in group 3 started treatment with 2,5HD on day 1; group 2 started treatment on day 14. On day 42 treatment was stopped in both groups; group 1 (control group) was never exposed to 2,5-HD. One week later (day 49) the females in all three groups were housed permanently with untreated proven breeder males (one male per female). Breeding cages were checked daily for the presence of newly born mice, which were sexed, counted, examined for gross abnormalities and discarded (in mice, the latter procedure maximizes the number of young born to each mother [8,14]). During the first 10 weeks litter weights were also recorded.
Statistical methods Body and organ weights, protein and DNA content and electromyographic parameters were analyzed by means of the non-parametric Mann and Whitney's Utest. An analysis of linear regression was performed on the results of the fertility study. The BMDP statistical package was run on an IBM-6660 mainframe computer.
Results
Treated mice grew more slowly than controls: the difference in body weight was approximately 10% after four weeks of treatment (P < 0.01) and approximately 14% after 6 weeks (P < 0.0001) (see Table I). During the mating period, which lasted 330 days, some deaths occurred: two dams in the control group and one male (which was not replaced) in the 4-week treatment group.
7 7
13 17
1 Control Treated
Control Treated
Control Treated
4
6
32.4 4- 1.7 27.9 4- 1.5"*
30.7 4- 1.9 27.7 4- 1.9"**
28.1 4- 1.8 28.0 4- 1.8
28.2 4- 1.5
Body weight (g)
*P < 0.05; **P < 0.01; ***P < 0.001.
6 10
7
No. of animals
0
Time (weeks)
10.0 4- 1.9 7.5 4- 1.2
8.9 4- 2.2 7.9 4- 2.0
8.8 4- 2.0 9.0 4- 1.2
8.2 4- 1.6
0.31 4- 0.05 0.27 4- 0.04
0.29 4- 0.07 0.29 4- 0.97
0.32 4- 0.06 0.32 4- 0.05
0.29 4- 0.05
1.27 4- 0.27 0.85 4- 0.21"*
1.01 4- 0.4 0.87 4- 0.3
1.26 4- 0.4 1.35 4- 0.5
0.91 4- 0.2
(mg/ovary)
(rag) (mg/g of body weight)
Protein
Weight of one ovary
126 4- 10 113 4- 15
111 4- 20 107 4- 13
140 4- 13 132 4- 12
110 4- 10
(/zg/mg tissue)
48.0 4- 6.6 38.0 4- 6.8*
56.0 4- 12.0 50.4 4- 9.7
58.8 4- 15.1 63.7 4- 16.2
60.0 4- 9.8
(rig/ovary)
DNA
4.8 4- 0.5 5.1 4- 0.4
6.3 4- 0.5 6.4 4- 0.6
6.6 4- 0.6 6.3 4- 0.3
7.4 4- 0.7
(/~g/mg tissue)
WEIGHT AND BIOCHEMICAL DATA (x" 4- SD) RELATIVE TO FEMALE MICE RECEIVING 1.5% 2,5-HEXANEDIONE IN THEIR D R I N K I N G WATER AS COMPARED WITH CONTROLS
TABLE I
'13 T A B L E II M O R P H O M E T R I C D A T A (NO, O F O O C Y T E S / A N I M A L ) (x 4. S.D.) RELATIVE TO F E M A L E MICE R E C E I V I N G 1.5% 2,5-HEXANEDIONE IN D R I N K I N G W A T E R AS C O M P A R E D W I T H CONTROLS Time (weeks)
No. of animals
Total
Small oocytes
Medium oocytes
Large oocytes
1 Control Treated
6 7
4248 4- 792 3942 4. 190
3390 4. 827 2990 4. 354
800 4. 163 706 4. 129
58 4. 12 61 4. 17
4
Control Treated
4 7
4184 4. 399 3428 4. 700
3179 4. 153 2566 4. 597
884 4- 290 746 4. 131
120 -4- 12 111 4. 23
6
Control Treated
6 9
3612 4. 758 2685 ± 498
3016 4. 700 2243 4. 503
540 4. 86 408 4. 95**
55 4. 27 40 4. 20
* * P < 0.01. a
20
10
0
_0
30 v
O ¢..
20
"I O"
ila-
10
0 20
10
In 012
3 4 5 6 7 8 9 10111213 No. of deliveries/fernole
Fig. 1. Frequency distribution histograms showing how the total number of litters delivered during the whole fertile life by female mice is affected by treatment with 2,5-hexanedione. (a) control, (b) 4-week treatment, (c) 6-week treatment.
0-32 a 33-52 53-72 73-92 93-112 113-132 133-152 153-172 173-192 193-212 213-232 233-252 253-272 273-292 293-312
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
11.4 13.1 12.1 11.4 11.6 9.8 9.6 7.1 6.2 5.7 4.7 2.5 8.0 1.0 4.5
4- 2.2 4- 1.8 4- 2.7 4- 3.6 4- 2.7 4- 2.7 4- 3.5 4- 2.7 4- 3.5 4- 3.4 4- 2.2 4- 2.1 4- 4.0 4- 0 4- 3.5
13 14 12 9 11 11 10 8 9 7 8 2 2 2 2
11.8 11.8 13.0 11.0 11.6 10.5 8.3 6.6 4.9 5.0 4.7 3.5 2.2 2.5 6.0
44444444444444-
2.9 2.4 2.6 2.8 2.6 2.8 2.8 4.5 3.0 3.1 2.5 1.4 0.7 0.5
Mean litter size 4- S.D.
Mean litter size 4- S.D.
No. of litters
4-week treatment
Control
13 14 14 13 15 11 14 8 11 8 4 6 6 2 1
No. of litters
aThe first interval is extended to day 32 to include the first wave of deliveries, which spanned from day 19 to 32.
Days after mating
Interval number
11.6 11.9 10.9 11.2 9.8 8.6 5.6 4.8 3.4 3.7 2.3 3.0 1.5 2.0
4444± 44444444-
2.4 2.1 3.3 2.8 2.8 3.6 3.3 3.5 2.0 2.2 1.7 1.4 0.7
Mean litter size 4- S.D.
6-week treatment
13 13 13 12 10 10 10 9 11 4 6 2 2 1
No. of litters
MEAN NUMBER OF NEWBORNS PER LITTER (LITTER SIZE), A N D NUMBER OF LITTERS BORN D U R I N G 20-DAY INTERVALS TO C O N T R O L AND TREATED MICE
TABLE III
45
Electromyography Mice receiving 1.5% 2,5-HD in their drinking water for up to six weeks did not show clinical symptoms of polyneuropathy, but electromyographic tests, performed on the tail nerves, revealed significant (P < 0.01) alterations in motor conduction velocity (-23%) and in distal latency time (+ 10%) after 5 weeks of treatment (see Ref. 3 for the meaning of these alterations in the diagnosis of polyneuropathy).
Biochemical studies The pooled results of two experiments are shown in Table I. In the first, 10 treated and 6 control mice were killed after 4 and 6 weeks of treatment; in the second, 7 treated and 7 control mice were killed after 1 and 4 weeks of treatment and 7 at zero time. At no time interval was the ovarian weight significantly altered in the treated animals, considering both absolute and relative (to body weight) values; protein and DNA content/mg of tissue were also not significantly altered. After 6 weeks of treatment the protein content/ovary showed a decrease of 31% (P < 0.01) and the DNA content/ovary a decrease of 21% (P < 0.05).
Morphology and morphometry Light microscope examination of ovarian sections did not reveal significant or consistent alterations in the histological structure of the organ in animals exposed to 2,5-HD (data not shown). The results of the morphometric study are reported in Table II. The values for the total number of oocytes and for oocytes belonging to each of the three categories were in all instances lower in the treated than in the control mice (with a single exception), but the difference reached statistical significance only for medium (growing) oocytes after 6 weeks (-25%, P < 0.01).
Effects on fertility Pups in all litters appeared normal by gross observation. Medium litter weight (g ± S.D.) during the first 10 weeks of mating was 19.9 4- 3.6 in controls, 20.0 4- 4.0 in the 4-week treatment group and 19.2 4- 3.9 in the 6-week group. Neither of these differences from controls were statistically significant. Medium pup weight was 1.63 4- 0.16 in the control group, 1.66 4- 0.20 in the 4-week treatment group and 1.67 ± 0.23 in the 6-week group. Figure 1 shows that while control mice had more frequently 10-11 deliveries, the 4-week treated ones had a maximum frequency of 9 deliveries and the 6-week treated group had 8-9. As the highest frequency of deliveries clustered around intervals of 20 days, in order to facilitate comparisons between groups, fertility data were subdivided into such intervals. Table III shows the mean litter size and the number of litters born to dams of the three groups during each interval. Both of these parameters decrease with time and this decrease appears to be slightly steeper in the 6-week treatment group than in the other two. The last delivery was recorded in the control group after 302 days of mating (410 days of age), in the 4-week treatment group after 294 days of mating and in the 6-week group after 278 days of mating. Another way of analyzing the data was based on the observation that no dif-
46 TABLE IV NUMBER OF NEWBORNS PER LITTER PRODUCED DURING THE FIRST 92 DAYS OF MATING BY FEMALE MICE PREVIOUSLY TREATED FOR VARIOUS PERIODS OF TIME WITH 2,5-HEXANEDIONE (NUMBER OF LITTERS IN PARENTHESES) Duration of treatment (weeks)
Litter size (x ± S.D.)
0 4 6 All
11.9 ±2.8 11.9 4- 2.8 11.4 ± 2.7 11.7 ± 2.7
(48) (54) (51) (153)
ference in litter size was evident between control and treated females for approximately the first 13 weeks o f continuous mating (Table IV). The average size o f litters delivered during this initial period (from zero to 92 days o f mating) was 11.7 4- 2.7 S.D. The n u m b e r o f pups p r o d u c e d at each delivery then began to decrease in a seemingly exponential fashion in both control and treated groups. Linear regression analysis was performed using data f r o m all deliveries occurring over the interval 9 3 - 3 0 2 mating days, after logarithmic transformation. Values and equations are reported in Table V; the regression lines are reported in Fig. 2 (together with the litter size values averaged over 20-day intervals). Test o f hypothesis on slope coefficients gave in all three cases P values smaller than 10 -4 (Fisher's F-test), meaning that in all three cases slope is significantly different from zero. The 6-week treatment regression line differed significantly from the control group (P < 10-5), which indicates a faster decrease in litter size with time in the treated animals. The 4-week regression line did not significantly differ from control.
Discussion This study shows only m i n o r effects o f a 6-week treatment with 1.5% 2,5-HD (in drinking water) on the reproductive system o f female mice. The treatment was suffi-
TABLE V PROGRESSIVE DECREASE IN THE NUMBER OF NEWBORNS PER LITTER PRODUCED DURING THE PERIOD 93-302 DAYS OF MATING. REGRESSION COEFFICIENTS AFTER LOGARITHMIC TRANSFORMATION OF DATA Duration of treatment (weeks)
Intercept
Slope ± S.E.
P(Fisher's F)
0 4 6
1.42 1.47 1.45
-3.7 x 10-3 4- 0.6 x 10-3 -4.2 x 10-3 4- 0.5 X 10-3 -5.1 X 10-3 4- 0.8 X 10-3*
< 104 < 10-4
39
Effects of 2,5-hexanedione on the ovary and fertility. An experimental study in mice G. Siracusa a, A. B a s t o n e b, M. Sbraccia b, L. Settimi b, C. M a l l o z z i b, E. M o n a c o c a n d N. Frontali b aDepartment of Public Health, Section of Anatomy & Cell Biology, 2nd University of Rome, blstituto Superiore di Sanita ', Rome and CLaboratory of Animal Sciences, ENEA, CRE Casaccia, Rome (Italy)
(Received January 28th, 1992; accepted June 24th, 1992)
Summary Sixty-day-old virgin female Swiss CD1 mice were treated with 1.5% 2,5-hexanedione in their drinking water; control mice received tap water; duration of treatment was either 4 or 6 weeks. Under these conditions the treated mice did not show any clinical symptoms although electromyography revealed some signs of polyneuropathy. Protein and DNA content per mg of ovarian tissue in treated mice were not significantly different from controls. Histological examination of ovarian sections at the light microscope level showed no significant alterations after exposure. A morphometric study revealed a statistically significant reduction in the number of growing oocytes after 6 weeks of treatment. For fertility studies three groups of 15 female mice each were treated for 0, 4 or 6 weeks as above and then permanently housed with untreated proven breeder male mice (one male per female); cages were checked daily for newly born mice. All litters appeared normal by gross examination. During the first 14 weeks of continuous mating the mean litter size (number of newborns per litter) remained about 1i.4 in all groups; this number subsequently began to decrease. Control and 4-week treatment regression curves did not differ statistically, while the slope of the 6-week line was significantly steeper, indicating a faster decrease in litter size over time and a shortening of fertile life in the latter group of treated females.
Key words: 2,5-Hexanedione; n-Hexane; Ovary; Fertility; Reproductive toxicology
Introduction n-Hexane has several applications in industry as a solvent, especially in glues, varnishes, cements and inks and is also used to extract oils from various seeds. Persons exposed to the solvent in poorly ventilated shoe and leather workshops and factories in Japan, Italy, France and Spain have developed severe forms of polyneuropathy; the employees were mainly women [1,2]. This neurologic condition has been reproduced in experimental animals treated with pure n-hexane (rat) or with its neurotoxic metabolite 2,5-hexanedione (cat, rat and mouse) [3]. A largely accepted Correspondence to." G. Siracusa, Dipartimento di Sanita' Pubblica e Biologia Cellulare, II Universita' di Roma, Via O. Raimondo, 00173 Roma, Italy. 0300-483X/92/$05.00 © 1992 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
40 hypothesis regarding the mode of action of this metabolite is that it binds to some cytoskeletal or cytoskeleton-associated proteins, altering their function (for a review see Ref. 4). Treatment of male rats with 2,5-hexanedione (2,5-HD) [5] or with n-hexane [6] has been found to cause testicular alterations in addition to polyneuropathy; practically no information is, however, available on the experimental effects of these compounds on the female reproductive system. The present work is a contribution towards filling this gap. Fewer quantitative biological markers have been elaborated for female than for male reproductive toxicity studies; histological assessment of ovarian follicle number is one endpoint in the female system [7], another is the total reproductive capacity test elaborated by Generoso et al. [8]. In most cases such work has been performed with mice rather than rats as experimental animals, mainly because of their better characterized reproductive parameters. In our study, the dose and the timing of treatment of female mice with 2,5-HD were chosen in such a way as to produce mild signs of polyneuropathy, as revealed through electromyography in a preliminary experiment. Materials and methods
Animals and treatments Virgin female Swiss CDI mice (Charles River, Italy) were randomly assigned to experimental and control groups and housed in plastic cages (6-10 per cage) at a room temperature of21°C ± 1 with a 12-h dark/light cycle (60% humidity); they had free access to food (a standard Vogt Miller diet in pellets) and water. Treatment began after a 10-day period of acclimation, when the mice were 60 days old, with the addition of 1.5% (v/v) 2,5-HD (Fluka AG, Buchs, Switzerland, re-purified by reflux distillation) to the animals' drinking water; duration of treatment was either 4 or 6 weeks. In each experiment all animals began treatment on the same day, with the exception of the fertility studies, where all ended on the same day (see below). The mice were weighed at the start of treatment and then at weekly intervals. Electrophysiological studies One experimental and one control group were employed, each consisting of 14 mice. The animals were subjected to electromyography at the onset and at weekly intervals for up to 5 weeks. Motor conduction velocity and distal latency time were measured on the tail nerves according to Miyoshi and Goto [9]. Biochemical studies For each time interval considered in the biochemical and morphological studies one experimental and one control group were used, each consisting of 6-10 mice. At given intervals, treated and control mice were sacrificed by cervical dislocation and the ovaries quickly removed. The right ovary was placed in a watchglass containing a physiological saline solution and was freed from surrounding adipose and connective tissue under a dissection microscope. It was then quickly blotted, weighed and homogenized using a glass Elvejhem Potter apparatus (Teflon pestle) in 10 ml of saline buffer (NaC1 2 M, Na2HPO4 0.05 M, EDTA 2 x 10-3 M, pH 7.4) per
41 g of tissue. The homogenate was assayed for protein [10] with BSA as a standard and for DNA [11]. Measurements were performed with a Zeiss PM7 spectrophotometer and with a Perkin Elmer LS-3 fluorescence spectrometer, respectively.
Morphological studies Light microscopic observation and oocyte morphometry were performed on left ovaries fixed in Bouin's medium, embedded in paraffin, serially sectioned at 6 #m and stained with haematoxylin and eosin. Oocytes were classified according to cellular diameter in three categories: small (resting oocytes, 70 #m) [12]. The small oocytes were counted in every twelfth section, the medium and large in every sixth; nucleoli were used as markers for all three categories of oocytes. The total number of oocytes per ovary was calculated according to Abercrombie [13]; all counts were made on one ovary and doubled to express the total number of oocytes per animal.
Fertility studies In order to study the effect on total reproductive capacity of a 4- or 6-week treatment with 2,5-HD, 8-week-old virgin female mice were randomly divided into three groups (each consisting of 15 mice). Animals in group 3 started treatment with 2,5HD on day 1; group 2 started treatment on day 14. On day 42 treatment was stopped in both groups; group 1 (control group) was never exposed to 2,5-HD. One week later (day 49) the females in all three groups were housed permanently with untreated proven breeder males (one male per female). Breeding cages were checked daily for the presence of newly born mice, which were sexed, counted, examined for gross abnormalities and discarded (in mice, the latter procedure maximizes the number of young born to each mother [8,14]). During the first 10 weeks litter weights were also recorded.
Statistical methods Body and organ weights, protein and DNA content and electromyographic parameters were analyzed by means of the non-parametric Mann and Whitney's Utest. An analysis of linear regression was performed on the results of the fertility study. The BMDP statistical package was run on an IBM-6660 mainframe computer.
Results
Treated mice grew more slowly than controls: the difference in body weight was approximately 10% after four weeks of treatment (P < 0.01) and approximately 14% after 6 weeks (P < 0.0001) (see Table I). During the mating period, which lasted 330 days, some deaths occurred: two dams in the control group and one male (which was not replaced) in the 4-week treatment group.
7 7
13 17
1 Control Treated
Control Treated
Control Treated
4
6
32.4 4- 1.7 27.9 4- 1.5"*
30.7 4- 1.9 27.7 4- 1.9"**
28.1 4- 1.8 28.0 4- 1.8
28.2 4- 1.5
Body weight (g)
*P < 0.05; **P < 0.01; ***P < 0.001.
6 10
7
No. of animals
0
Time (weeks)
10.0 4- 1.9 7.5 4- 1.2
8.9 4- 2.2 7.9 4- 2.0
8.8 4- 2.0 9.0 4- 1.2
8.2 4- 1.6
0.31 4- 0.05 0.27 4- 0.04
0.29 4- 0.07 0.29 4- 0.97
0.32 4- 0.06 0.32 4- 0.05
0.29 4- 0.05
1.27 4- 0.27 0.85 4- 0.21"*
1.01 4- 0.4 0.87 4- 0.3
1.26 4- 0.4 1.35 4- 0.5
0.91 4- 0.2
(mg/ovary)
(rag) (mg/g of body weight)
Protein
Weight of one ovary
126 4- 10 113 4- 15
111 4- 20 107 4- 13
140 4- 13 132 4- 12
110 4- 10
(/zg/mg tissue)
48.0 4- 6.6 38.0 4- 6.8*
56.0 4- 12.0 50.4 4- 9.7
58.8 4- 15.1 63.7 4- 16.2
60.0 4- 9.8
(rig/ovary)
DNA
4.8 4- 0.5 5.1 4- 0.4
6.3 4- 0.5 6.4 4- 0.6
6.6 4- 0.6 6.3 4- 0.3
7.4 4- 0.7
(/~g/mg tissue)
WEIGHT AND BIOCHEMICAL DATA (x" 4- SD) RELATIVE TO FEMALE MICE RECEIVING 1.5% 2,5-HEXANEDIONE IN THEIR D R I N K I N G WATER AS COMPARED WITH CONTROLS
TABLE I
'13 T A B L E II M O R P H O M E T R I C D A T A (NO, O F O O C Y T E S / A N I M A L ) (x 4. S.D.) RELATIVE TO F E M A L E MICE R E C E I V I N G 1.5% 2,5-HEXANEDIONE IN D R I N K I N G W A T E R AS C O M P A R E D W I T H CONTROLS Time (weeks)
No. of animals
Total
Small oocytes
Medium oocytes
Large oocytes
1 Control Treated
6 7
4248 4- 792 3942 4. 190
3390 4. 827 2990 4. 354
800 4. 163 706 4. 129
58 4. 12 61 4. 17
4
Control Treated
4 7
4184 4. 399 3428 4. 700
3179 4. 153 2566 4. 597
884 4- 290 746 4. 131
120 -4- 12 111 4. 23
6
Control Treated
6 9
3612 4. 758 2685 ± 498
3016 4. 700 2243 4. 503
540 4. 86 408 4. 95**
55 4. 27 40 4. 20
* * P < 0.01. a
20
10
0
_0
30 v
O ¢..
20
"I O"
ila-
10
0 20
10
In 012
3 4 5 6 7 8 9 10111213 No. of deliveries/fernole
Fig. 1. Frequency distribution histograms showing how the total number of litters delivered during the whole fertile life by female mice is affected by treatment with 2,5-hexanedione. (a) control, (b) 4-week treatment, (c) 6-week treatment.
0-32 a 33-52 53-72 73-92 93-112 113-132 133-152 153-172 173-192 193-212 213-232 233-252 253-272 273-292 293-312
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
11.4 13.1 12.1 11.4 11.6 9.8 9.6 7.1 6.2 5.7 4.7 2.5 8.0 1.0 4.5
4- 2.2 4- 1.8 4- 2.7 4- 3.6 4- 2.7 4- 2.7 4- 3.5 4- 2.7 4- 3.5 4- 3.4 4- 2.2 4- 2.1 4- 4.0 4- 0 4- 3.5
13 14 12 9 11 11 10 8 9 7 8 2 2 2 2
11.8 11.8 13.0 11.0 11.6 10.5 8.3 6.6 4.9 5.0 4.7 3.5 2.2 2.5 6.0
44444444444444-
2.9 2.4 2.6 2.8 2.6 2.8 2.8 4.5 3.0 3.1 2.5 1.4 0.7 0.5
Mean litter size 4- S.D.
Mean litter size 4- S.D.
No. of litters
4-week treatment
Control
13 14 14 13 15 11 14 8 11 8 4 6 6 2 1
No. of litters
aThe first interval is extended to day 32 to include the first wave of deliveries, which spanned from day 19 to 32.
Days after mating
Interval number
11.6 11.9 10.9 11.2 9.8 8.6 5.6 4.8 3.4 3.7 2.3 3.0 1.5 2.0
4444± 44444444-
2.4 2.1 3.3 2.8 2.8 3.6 3.3 3.5 2.0 2.2 1.7 1.4 0.7
Mean litter size 4- S.D.
6-week treatment
13 13 13 12 10 10 10 9 11 4 6 2 2 1
No. of litters
MEAN NUMBER OF NEWBORNS PER LITTER (LITTER SIZE), A N D NUMBER OF LITTERS BORN D U R I N G 20-DAY INTERVALS TO C O N T R O L AND TREATED MICE
TABLE III
45
Electromyography Mice receiving 1.5% 2,5-HD in their drinking water for up to six weeks did not show clinical symptoms of polyneuropathy, but electromyographic tests, performed on the tail nerves, revealed significant (P < 0.01) alterations in motor conduction velocity (-23%) and in distal latency time (+ 10%) after 5 weeks of treatment (see Ref. 3 for the meaning of these alterations in the diagnosis of polyneuropathy).
Biochemical studies The pooled results of two experiments are shown in Table I. In the first, 10 treated and 6 control mice were killed after 4 and 6 weeks of treatment; in the second, 7 treated and 7 control mice were killed after 1 and 4 weeks of treatment and 7 at zero time. At no time interval was the ovarian weight significantly altered in the treated animals, considering both absolute and relative (to body weight) values; protein and DNA content/mg of tissue were also not significantly altered. After 6 weeks of treatment the protein content/ovary showed a decrease of 31% (P < 0.01) and the DNA content/ovary a decrease of 21% (P < 0.05).
Morphology and morphometry Light microscope examination of ovarian sections did not reveal significant or consistent alterations in the histological structure of the organ in animals exposed to 2,5-HD (data not shown). The results of the morphometric study are reported in Table II. The values for the total number of oocytes and for oocytes belonging to each of the three categories were in all instances lower in the treated than in the control mice (with a single exception), but the difference reached statistical significance only for medium (growing) oocytes after 6 weeks (-25%, P < 0.01).
Effects on fertility Pups in all litters appeared normal by gross observation. Medium litter weight (g ± S.D.) during the first 10 weeks of mating was 19.9 4- 3.6 in controls, 20.0 4- 4.0 in the 4-week treatment group and 19.2 4- 3.9 in the 6-week group. Neither of these differences from controls were statistically significant. Medium pup weight was 1.63 4- 0.16 in the control group, 1.66 4- 0.20 in the 4-week treatment group and 1.67 ± 0.23 in the 6-week group. Figure 1 shows that while control mice had more frequently 10-11 deliveries, the 4-week treated ones had a maximum frequency of 9 deliveries and the 6-week treated group had 8-9. As the highest frequency of deliveries clustered around intervals of 20 days, in order to facilitate comparisons between groups, fertility data were subdivided into such intervals. Table III shows the mean litter size and the number of litters born to dams of the three groups during each interval. Both of these parameters decrease with time and this decrease appears to be slightly steeper in the 6-week treatment group than in the other two. The last delivery was recorded in the control group after 302 days of mating (410 days of age), in the 4-week treatment group after 294 days of mating and in the 6-week group after 278 days of mating. Another way of analyzing the data was based on the observation that no dif-
46 TABLE IV NUMBER OF NEWBORNS PER LITTER PRODUCED DURING THE FIRST 92 DAYS OF MATING BY FEMALE MICE PREVIOUSLY TREATED FOR VARIOUS PERIODS OF TIME WITH 2,5-HEXANEDIONE (NUMBER OF LITTERS IN PARENTHESES) Duration of treatment (weeks)
Litter size (x ± S.D.)
0 4 6 All
11.9 ±2.8 11.9 4- 2.8 11.4 ± 2.7 11.7 ± 2.7
(48) (54) (51) (153)
ference in litter size was evident between control and treated females for approximately the first 13 weeks o f continuous mating (Table IV). The average size o f litters delivered during this initial period (from zero to 92 days o f mating) was 11.7 4- 2.7 S.D. The n u m b e r o f pups p r o d u c e d at each delivery then began to decrease in a seemingly exponential fashion in both control and treated groups. Linear regression analysis was performed using data f r o m all deliveries occurring over the interval 9 3 - 3 0 2 mating days, after logarithmic transformation. Values and equations are reported in Table V; the regression lines are reported in Fig. 2 (together with the litter size values averaged over 20-day intervals). Test o f hypothesis on slope coefficients gave in all three cases P values smaller than 10 -4 (Fisher's F-test), meaning that in all three cases slope is significantly different from zero. The 6-week treatment regression line differed significantly from the control group (P < 10-5), which indicates a faster decrease in litter size with time in the treated animals. The 4-week regression line did not significantly differ from control.
Discussion This study shows only m i n o r effects o f a 6-week treatment with 1.5% 2,5-HD (in drinking water) on the reproductive system o f female mice. The treatment was suffi-
TABLE V PROGRESSIVE DECREASE IN THE NUMBER OF NEWBORNS PER LITTER PRODUCED DURING THE PERIOD 93-302 DAYS OF MATING. REGRESSION COEFFICIENTS AFTER LOGARITHMIC TRANSFORMATION OF DATA Duration of treatment (weeks)
Intercept
Slope ± S.E.
P(Fisher's F)
0 4 6
1.42 1.47 1.45
-3.7 x 10-3 4- 0.6 x 10-3 -4.2 x 10-3 4- 0.5 X 10-3 -5.1 X 10-3 4- 0.8 X 10-3*
< 104 < 10-4
