Effects of Morpholine on Fertility

Morpholine is an organic chemical compound having the chemical formula O(CH2CH2)2NH.With both amine and ether functional groups.Because of the amine group, morpholine is a base.It is used as a component in fungicides and as a chemical emulsifier in the process of waxing fruit, therefore the possibility of food contamination is probable. Exposure to morpholine causes toxic hazards,

to people with impaired kidney function, as well as chronic toxic hazards of gene mutations, cancer, neurotoxicity, bone pathology, and reproductive effects.

there is an association of decreasing total fertility rate with increasing morpholine levels ,decreased testosterone concentrations and a significant decline in sperm motility and reproductive toxicity of morpholine.Thus,the present study investigates the effect of both short term (2-week) and long term (4-week) exposure to morpholine on fertility .

EFFECTS OF MORPHOLINE ON FERTILITY  

By Mohammad Ali Kavianpour, M.D

Kermanshah University of Medical Sciences, November, 2008

SUMMARY

Full-grown female mice were allowed  free access to water containing 0, 10, 20 and 30 ppm morpholine for either 2 weeks or 4 weeks. The effect of morpholine exposure on fertility was assessed in terms of the numbers of pregnant mice, implantations, viable fetuses, and resorptions.

Exposure to morpholine for 2 weeks did not have much effect on fertility, although there was a significant increase in the relative ovary weights and a decrease in the embryo weights in mice exposed to 30 ppm .However, exposure  for 4 weeks resulted in a significant reduction in the percentage of pregnancies at all concentrations used.

Mice exposed to 20 and 30 ppm showed a significant increase in relative ovary weights and a decrease in the number of viable fetuses. Furthermore, exposure to 30 ppm for 4 weeks resulted in a significant decrease in number of implantations. These results indicate that long-term exposure of female mice to morpholine causes adverse effects on the reproductive system and fertility.

INTRODUCTION

Morpholine is an organic chemical compound having the chemical formula O(CH 2CH2)2NH.With both amine and ether functional groups.Because of the amine group, morpholine is a base.It is used as a component in fungicides and as a chemical emulsifier in the process of waxing fruit, therefore the possibility of food contamination is probable. Exposure to morpholine causes toxic hazards,

to people with impaired kidney function, as well as chronic toxic hazards of gene mutations, cancer, neurotoxicity, bone pathology, and reproductive effects.

there is an association of decreasing total fertility rate with increasing morpholine levels ,decreased testosterone concentrations and a significant decline in sperm motility and reproductive toxicity of morpholine.Thus,the present study investigates the effect of both short term (2-week) and long term (4-week) exposure to morpholine on fertility.

MATERIALS AND METHODS

Eighty mature  female  mice (50 days old) were used in this study.They were raised in the animal house under a controlled temperature of 22±1.0°C on a 12-hr light/dark cycle. Standard Food and water were supplied. Since both the diet and water had low levels of morpholine,calculations of morpholine intake are based exclusively on the morpholine added to the drinking water. Mice were exposed for 2 weeks or 4 weeks to different concentrations (10, 20, and 30 ppm) of morpholine  dissolved in tap water. For either exposure time, mice were randomly divided into four groups . The first group served as the reference and the animals were allowed free access to  water without any added morpholine. The other three groups were allowed  free access to  water containing either 10, 20, or 30 ppm morpholine.

Animals were observed daily from the first day of exposure to morpholine for clinical signs of toxicity. Their water consumption was measured every day and the body weights every week.The effect of morpholine ingestion on the occurrence of implantation was estimated in the mice and in their control counterparts after the appropriate time of exposure. Treated mice and their control counterparts were divided randomly into groups of two animals each and housed with a mature untreated male of proven fertility for ten days. During this period, at least two estrous cycles should have elapsed. One week after the removal of untreated males, the treated females and their control counterparts were killed by cervical dislocation under light ether anesthesia. The following measurements were recorded: number of pregnant females, number of implantations, number of viable fetuses, and number of resorptions. Furthermore, the maternal body, uterus, and ovary weights were measured, and the embryo weights were also recorded.

RESULTS

Exposure levels and toxicity of morpholine:

The actual doses that the animals received based on the water consumption per kg body weight per day were 0.95 – 2.77 mg morpholine for the three groups after 2 weeks of exposure and 0.58 – 1.88 mg for the three groups after 4 weeks of exposure None of the animals in the 2-week exposure group showed any clinical signs of toxicity. However, for the 4-week exposure group, 1 animal out of

10, 2 animals out of 10, and 2 animals out of 10 died in the groups exposed to 10, 20, and 30 ppm morpholine, respectively.

1)Average water consumption of female mice exposed to morpholine for 2 weeks :

Treatment &n bsp;       No. of animals     Water consumption (mL/day)     Dose (mg/kg/day)

    0       &n bsp;              10       & nbsp;          ;      3.39 ± 0.05                  &nbs p;         &n bsp;       0

 (10 ppm)             10       & nbsp;          ;      4.21 ± 0.07                  &nbs p;                 0.95 ± 0.017

 (20 ppm)             10       & nbsp;          ;      4.12 ± 0.49                  &nbs p;               &n bsp; 1.84 ± 0.22

 (30 ppm)             10       & nbsp;          ;      3.31 ± 0.37                  &nbs p;             &n bsp;   2.77 ± 0.303

2)Average water consumption of female mice exposed to morpholine for 4 weeks:

Treatment &n bsp;        No. of animals       Water consumption (mL/day)      Dose of (mg/kg/day)

   0       &n bsp;               10       & nbsp;          ;         2.71 ± 0.09                  &nbs p;         &n bsp;         0

 (10 ppm)          &nb sp;  9      ;         &nb sp;         & nbsp;  2.42 ± 0.89                  &nbs p;         &n bsp;         0.58 ± 0.080

 (20 ppm)              8       &n bsp;                    2.27 ± 0.12                  &nbs p;         &n bsp;         1.27 ± 0.111

 (30 ppm)              8       &n bsp;                    2.25 ± 1.1        & nbsp;          ;         &nb sp;         & nbsp; 1.88 ± 0.073

Effect of morpholine on fertility:

There were no significant differences between the control and the morpholine treated groups regard to the number of females becoming pregnant nor on the number of implantations. The number of viable fetuses was slightly lower in the morpholine treated groups.There were significant decreases in the percentage of pregnant mice in the morpholine treated groups in comparison to their control counterparts. Furthermore, exposure morpholine resulted in a decrease in the number of implantations at all concentrations used but the decrease was statistically significant only in the group exposed to 30 ppm. There was also a significant reduction in the number of viable fetuses in mice exposed to 20 and 30 ppm.

Effect of morpholine on maternal organ weights and embryo weights:

Ingestion of 30 ppm morpholine for 2 weeks resulted in a significant increase in the relative ovary weight. There was also a statistically significant increase in the embryo weights of animals exposed to 20 and 30 ppm , in comparison to the  control counterparts.

There were no significant differences in the uterine weights of animals exposed to morpholine for 2 weeks in comparison to their controls.Ingestion of 20 and 30 ppm morpholine for 4 weeks also resulted in a significant increase in the relative ovary weights.Animals exposed to 30 ppm  showed a significant increase in the embryo weights in comparison to the control counterparts.No significant differences were observed in the uterine weights of animals  exposed to morpholine for 4 weeks in comparison to their controls.

3)Effect of 2-week exposure to morpholine on fertility of female mice

Treatment &n bsp;              No of pregnant females      implantations   & nbsp; viable fetuses      resorptions/ implantations

   0       &n bsp;                       10/10             &nbs p;         &n bsp;        10.1 ± 0.50          10.0 ± 0.60               1/100

 (10 ppm)                     10/10      &nbs p;         &n bsp;               9.7 ± 0.64             9.3 ± 0.54                 1/95

 (20 ppm)                     10/10      &nbs p;         &n bsp;               9.6 ± 0.82             9.2 ± 0.92                 2/94

 (30 ppm)                     10/10      &nbs p;         &n bsp;              9 .4 ± 0.35             9.1 ± 0.25                 3/92

4)Effect of 4-week exposure to morpholine on fertility of female mice

Treatment &n bsp;            No. of pregnant  females      implantations    viable fetuses     resorptions/ implantations

   0       &n bsp;                   9/10 (90%)         &nbs p;         &n bsp;    &nbs p; 7.34 ± 0.86        7.34 ± 0.83                  0/66

 (10 ppm)                  6/9 (66%†)      &nb sp;         & nbsp;         5.51 ± 1.02        5.02 ± 1.15                  3/33

 (20 ppm)                  4/8 (50%‡)      &nb sp;         & nbsp;         5.26 ± 1.11        4.04 ± 0.73                  5/21

 (30 ppm)                  5/8 (62%†)      &nb sp;         & nbsp;         5.09 ± 0.33        4.25 ± 0.87       &nb sp;         < /span>4/20

5)Effect of 2-week exposure to morpholine on maternal organ weights and embryo weights

Treatment &n bsp;   females  Bwt on day of sacrifice(g) Ovary mg/10gBwt   Uterus (mg/10gBwt)    Embryo (g)

   0    & nbsp;            10   &nb sp;         45.78 ± 1.20                  &nbs p;      10.19 ± 0.69             201.6 ± 6.1        & nbsp;        0.748 ± 0.042

 (10 ppm)       10         &nb sp;   45.08 ± 1.80                  &nbs p;       9.83 ± 0.59              204.8 ± 4.5        & nbsp;        0.815 ± 0.092

 (20 ppm)       1 0        &nb sp;    42.59 ± 6.03                  &nbs p;       9.98 ± 0.69              209.6 ± 12        &n bsp;        1.015 ± 0.066

 (30 ppm)       1 0        &nb sp;    36.23 ± 1.10                  &nbs p;       12.55 ± 0.65            183.9 ± 7.6        & nbsp;        1.177 ± 0.058

6)Effect of 4-week exposure to morpholine on maternal organ weights and embryo weights

Treatment &n bsp;   females  Bwt on day of sacrifice(g) Ovary mg/10gBwt   Uterus (mg/10gBwt)    Embryo (g)

    0    & nbsp;           10       &nbs p;     45.22 ± 1.54                  &nbs p;    11.5 ± 1.28                142.9 ± 8.70                 0.814 ± 0.039

 (10 ppm)       9          &nbs p;     4 2.71 ± 2.41                       11.3 ± 1.26                123.2 ± 16.0                 0.648 ± 0.100

 (20 ppm)       8          &nbs p;     3 5.03± 0.95                       &n bsp;17.4 ± 2.29                1 32.4 ± 17.0                 0.633 ± 0.084

 (30 ppm)       8         &nbs p;      3 9.17 ± 2.01          &nb sp;         & nbsp; 14.3 ± 0.34                151.6 ± 12.0                1.159 ± 0.047

DISCUSSION

This research investigated the effect on fertility of 2-week and 4-week exposureof mature  female mice to 10, 20, and 30 ppm morpholine administered in drinking water.The animal model used in this study has been used previously in several studies to assess the adverse effects of different compounds It was shown that exposing mature  female mice to morpholine in drinking water for 2 weeks had no significant effect on their fertility. However, there was a statistically significant increase in the relative ovary weights at 30 ppm and in embryo   weights at 20 and 30 ppm. Exposure of mice for 4 weeks resulted in a significant increase in the relative ovary weights at both 20 ppm and 30 ppm morpholine and in embryo weights at 30 ppm. Thus short-term and chronic exposure to morpholine results in increased embryo weights at the highest dose level of 30 ppm. The increase in ovarian weights in mice exposed to morpholine needs to be clarified by histological studies. This will help to explain whether the weight increase in the ovaries observed in this study is due to hyperplasia or hypertrophy of a specific tissue compartment in this organ. Any increase in the weight of reproductive organs is under hormonal control. The results, therefore, suggest a disturbance of the reproductive endocrine functions, possibly with multiple sites of toxicity along the hypothalamic pituitary ovarianuterine axis.

The main finding of the present study was the significant reduction in the occurrence of pregnancy in mice exposed to morpholine for 4 weeks. This decrease may be due to alteration of the reproductive endocrine functions leading to decreased secretion of progesterone which is needed for endometrial alteration at the time of implantation and is necessary for successful pregnancy. This may also explain the significant decrease in the number of implantations resulting in a decrease in the number of viable fetuses. We can also investigating the effect of morpholine exposure on serum progesterone levels.Administration of morpholine, at 20 ppm, resulted in lower egg weights and lengths and in a 10% reduction of the weights of dayone hatchlings in comparison to their control counterparts.

Keywords:
Female mice,  Fertility, Reproductive effects, Morpholine

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