Ameliorative role of amino acids on fluoride-induced alterations in mice (part ii): ovarian and uterine nucleic acid metabolism

FLUORIDE 31 (3) 1998, pp 143-148	International Society for Fluoride Research	Table of Contents

AMELIORATIVE ROLE OF AMINO ACIDS ON FLUORIDE-INDUCED
ALTERATIONS IN MICE (PART II): OVARIAN AND
UTERINE NUCLEIC ACID METABOLISM

Dhruva Patel and N J Chinoy Ahmedabad, India

SUMMARY: Sodium fluoride (5 mg/kg body weight) was effective from the 45th day of treatment in causing a significant decline in DNA and RNA levels of mice ovary and uterus, indicating alterations in nucleic acid and protein metabolism in these organs. The oestrus cycle was irregular with prolonged duration of the diestrus stage which in turn severely affected the fertility rate in treated mice.

The administration of amino acids glycine and glutamine, individually and in combination along with NaF, helped in maintaining the status quo of all parameters as compared to control, thus elucidating their ameliorative role.

Key words: Amino acids; DNA; Fertility; Mice; RNA; Sodium fluoride.

Department of Zoology, School of Sciences, Gujarat University, Ahmedabad 380 009, India. Correspondence to Professor N J Chinoy.
This study is Part II of the earlier experiment reported in Fluoride 29 (4) 217-226 1996.

INTRODUCTION

Tsutsui et al described genotoxic effects of fluoride.¹ NaF has been reported to cause an increase in the frequency of Sister Chromatid Exchange (SCE) in human population of endemic areas of North Gujarat.²

Increased feeding of fluoride to animals and humans raises the fluoride concentration in maternal and fetal blood.³ Administration of 150-300 mg F^–/kg body weight blocked gonadotropin stimulation of rabbit ovary.4 Hanley et al reported maternal weight loss and fetotoxic effects of fluoride in white rabbits and rats exposed to 225 ppm of fluoride.⁵ Messer et al have attempted to link infertility and fluorosis.⁶ Reports from our laboratory have clearly elucidated alterations in carbohydrate and oxidative metabolisms and altered functions of some vital and reproductive organs in fluoridated female mice.^7-9 However, the effects in the ovary and uterus due to fluoride are not yet fully explored.

In the light of earlier data the purpose of the present investigation was to evaluate the effects of fluoride on nucleic acid and protein levels in the ovary and uterus and the fertility impairment in mice under experimental fluorosis. The possible role of amino acids (glycine and glutamine) in the amelioration of fluoride induced toxicity was also investigated.

MATERIALS AND METHODS

The animals used, exposures, protein levels and experimental protocol are the same as described earlier by Chinoy and Patel.⁹ The protein levels used in this study are also the same as given in Chinoy and Patel.⁹

Nucleic Acids:
The deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) in the ovary and uterus were first extracted in trichloroacetic acid (TCA) and the RNA levels were determined using orcinol as the colouring reagent by the method of Mejboum.¹⁰ The resulting colour was read at 670 nm on a Systronics Colorimeter 106 and expressed as µ moles/100 mg fresh tissue weight.

The DNA levels were estimated by the method of Giles and Meyer¹¹ using diphenylamine as the colouring reagent and the OD of the resultant colour was read at 620 nm on a Systronics 106 Colorimeter and expressed as µ moles/100 mg fresh tissue weight.

Fertility Test: was assessed according to the WHO protocol MB-50.¹²

Cyclicity: The Oestrus cycle was recorded in normal and NaF treated mice, by observing the vaginal smear daily, for over a period of two months.

Statistics: For each biochemical estimation, a minimum of 6 to 8 replicates were carried out and the data was analyzed using Analysis of variance (ANOVA) followed by Scheff ’s test.

RESULTS

Deoxyribonucleic acid (DNA):
NaF treatment resulted in a significant suppression (P<0.001) of ovarian and uterine DNA levels. However, after administration of glycine and/or glutamine (group III, IV, V) the DNA levels were almost similar to those of control (Table 1), which implies a significant increase (P<0.001) as compared to the NaF treated group.

Ribonucleic acid (RNA):
NaF treatment (45, 60 days) caused significant (P<0.001) decline in ovarian and uterine RNA levels as compared to control, whereas, after administration of glycine and/or glutamine along with NaF (group III, IV, V) the ribonucleic acid levels were similar to those in the control mice (Table 2) which reveals a significant increase (P<0.001) as compared to the treated group.

DNA/RNA ratio:
The DNA/RNA ratio in uterus declined (P<0.05) after 45days and 60 days of treatment, whereas it increased (P<0.05) in the ovary. NaF treatment along with glycine and/or glutamine resulted in increase (P<0.05) in the DNA/RNA ratio in the uterus as compared to NaF treated group whereas in the ovary the ratio remained unaltered (Table 3).

RNA/Protein ratio:
A significant decline (P<0.001) in RNA/protein ratio occurred in the ovary after NaF treatment (45 and 60 days), while, no change was observed in uterus. After administration of glycine or glutamine along with NaF (group III and IV), the ratio was the same as in control (P<0.05). The levels were almost the same as control in group V in which animals were fed glycine and glutamine in combination with NaF (Table 4), implying a significant increase as compared to the NaF treated group.

Fertility rate :
Sodium fluoride treatment led to a significant decline (P<0.001) in fertility rate after 45 and 60 days of treatment. The fertility rate was less affected after ingestion of glycine or glutamine alone and in combination along with NaF (Table 5) than in NaF treatment alone (group II).

Cyclicity:
The cyclicity of NaF treated animals was disturbed. The cycles were irregular and prolonged. However, in groups III, IV and V regular cycles were observed but their duration was extended (Table 5).

TABLE 1A. DNA levels (µ moles/100mg fresh tissue weight)
in uterus and ovary of control and treated groups

Uterus Ovary

Groups 45 days 60 days 45 days 60 days

Control 886.34 ± 20.2 894.30 ± 18.1 1053 ± 36.3 1002.0 ± 21.2

NaF 484.79 ± 17.9^* 408.28 ± 16.6^* 658 ± 17.0^* 599.0 ± 18.2^*

NaF+Glycine 807.57 ± 12.2^* 907.72 ± 18.2^* 923 ± 16.3^* 976.0 ± 19.1^*

NaF + Glutamine 794.15 ± 19.9^* 865.28 ± 15.1^* 936 ± 19.9^* 941.44 ± 16.6^*

NaF+Glycine
+ Glutamine 907.12 ± 24.8^* 974.56 ± 14.2^* 1024 ± 32.6^* 1056.0 ± 21.9^*

Values are Mean + Std Error * P < 0.001

TABLE 1B. DNA ANOVA

Source of
variation SS df MSS f(cal) f(tab) P value

Uterus
Groups
Residual
1104058.00
20732.00
8
18
138007.00
1151.00
119.80

3.71

<0.001

Ovary
Groups
Residual
849933.60
41481.00
8
18
106241.00
2304.00
46.10
3.71
<0.001

SS = Sum of squares df = Degrees of freedom MSS = Mean sum of squares

TABLE 2A. RNA levels (µ moles/100 mg fresh tissue weight)
in uterus and ovary of control and treated groups

Uterus Ovary

Groups 45 days 60 days 45 days 60 days

Control 151.52 ± 6.2 149 ± 7.6 221.51 ± 11.1 208 ± 19.6

NaF 112.51 ± 5.9^** 99.21 ± 7.9^*** 104.19 ± 11.3^*** 98.86 ± 10.0^***

NaF+Glycine 130.93 ± 6.7^* 117.67 ± 8.2^** 152.86 ± 8.7^** 173.66 ± 12.1^***

NaF+Glutamine 119.13 ± 2.3^NS 113.48 ± 4.2^** 142.97 ± 10.2^** 168.71 ± 10.9^***

NaF+Glycine
+ Glutamine 134.8 ± 9.2^* 129.98 ± 7.6^*** 176 ± 8.6^** 188.82 ± 11.3^***

Values are Mean ± Std Error ^***P < 0.001 ^**P < 0.01 ^*P < 0.05

TABLE 2B. RNA ANOVA

Source of
variation SS df MSS f(cal) f(tab) P value

Uterus

Groups 8280.65 8 1035.00 5.57 3.71 <0.001

Residual 3342.00 18 185.00

Ovary

Groups 42369.30 8 5296.00 17.00 3.71 <0.001

Residual 5604.60 18 311.00

SS = Sum of squares df = Degrees of freedom MSS = Mean sum of squares

TABLE 3A. DNA/RNA ratio in uterus and ovary of control and treated groups

Uterus Ovary

Groups 45 days 60 days 45 days 60 days

Control 5.76 ± 0.4 5.90 ± 0.3 4.77 ± 1.1 4.54 ± 0.6

NaF 4.29 ± 0.3^* 4.17 ± 1.2^* 6.39 ± 0.3^* 6.08 ± 1.4^*

NaF+Glycine 6.38 ± 0.9^* 7.71 ± 0.6^* 6.23 ± 1.9^* 5.75 ± 0.9^*

NaF + Glutamine 7.10 ± 0.6^* 7.60 ± 0.1^* 6.71 ± 2.0^* 6.55 ± 1.3^*

NaF+Glycine
+ Glutamine 6.93 ± 0.4^* 7.35 ± 0.3^* 5.81 ± 1.4^* 5.60 ± 0.8^*

Values are Mean ± Std Error *P < 0.05

TABLE 3B. DNA/RNA Ratio ANOVA

Source of
variation SS df MSS f(cal) f(tab) P value

Uterus

Groups 62.55 8 7.82 7.28 3.26 <0.05

Residual 29.00 27 1.07

Ovary

Groups 16.07 8 2.01 4.17 3.71 <0.05

Residual 8.67 18 0.48

SS = Sum of squares df = Degrees of freedom MSS = Mean sum of squares

TABLE 4A. RNA/Protein^†† ratio in uterus and ovary of control and treated groups

Uterus Ovary

Groups 45 days 60 days 45 days 60 days

Control 10.53 ± 0.32 10.41 ± 2.10^NS 18.22 ± 0.90 19.6 ± 0.72

NaF 11.49 ± 0.61^NS 10.88 ± 1.00 ^NS 13.10 ± 1.21^** 13.49 ± 0.67^**

NaF+Glycine 10.10 ± 0.77^NS 8.94 ± 0.96 ^NS 14.14 ± 0.62^* 15.03 ± 0.62^*

NaF + Glutamine 8.57 ± 0.31^NS 8.85 ± 0.62 ^NS 12.96 ± 0.81^* 13.92 ± 1.62^*

NaF+Glycine
+ Glutamine 9.26 ± 0.40^NS 9.05 ± 0.91 ^NS 16.60 ± 1.30^** 16.20 ± 1.42^**

Values are Mean ± Std Error ^*P < 0.05 ^**P < 0.001
^††For protein levels in ovary and uterus see Part I (reference no. 9)

TABLE 4B. RNA/Protein Ratio ANOVA

Source of
variation SS df MSS f(cal) f(tab) P value

Uterus

Groups 30.29 8 3.78 1.86 3.71 NS

Residual 36.47 18 2.02

Ovary

Groups 143.37 8 17.92 7.57 3.71 <0.001

Residual 42.57 18 2.36

SS = Sum of squares df = Degrees of freedom MSS = Mean sum of squares
NS = Non significant

TABLE 5. Fertility rate (%) and implantation sitesof treated and control groups of mice

No. of females mated/
No. of females pregnant Fertility rate
† † (%) No. of
implantation sites Cyclicity

45 days 60 days 45 days 60 days 45 days 60 days 45 days 60 days

Control 10/10 10/10 100 100 12.8± 0.9 12.5± 0.7 Regular Regular

NaF 10/6 10/2 60 20 3.2± 0.5^** 1.5± 0.5^** Irregular Irregular

NaF+Glycine 10/9 10/9 90 90 8.5± 1.4^* 6.5± 0.3 Regular^† Regular^†

NaF+Glutamine 10/9 10/9 90 90 8.0± 1.3^* 7.0± 1.1^* Regular^† Regular^†

NaF+Glycine
+ Glutamine 10/10 10/10 100 100 9.0± 1.5^** 9.0± 1.2^** Regular^† Regular^†

  ^†The oestrus cycle was found to be regular but duration of cycle was extended.
  ^† †Females:Males used in ratio of 2:1.
  ^**P< 0.001   ^*P<0.01
  At least 3 sets done for each group and mean values are presented.

DISCUSSION

Several human conditions including aging, cancer and arteriosclerosis have been associated with DNA damage and its misrepair.¹³ Fluoride has been reported to cause depression in DNA and RNA synthesis in cultured cells.¹⁴ In the present study, the DNA and RNA concentrations in the ovary and uterus were significantly decreased, which could be due to a decrease in their synthesis or an alteration in their metabolism. Earlier reports have also revealed that fluoride ingestion in rabbits resulted in a decrease in DNA and RNA levels in the ovary¹⁵ in corroboration with the present data. The inhibition of DNA and RNA synthesis may result in delayed mitotic and meiotic cycles including chromosomal breakages.¹⁶ Studies on fluorotic human population in endemic areas of North Gujarat have shown an increase in frequency of sister chromatid exchange as compared to the control indicating that fluoride might have a genotoxic effect.²

The DNA/RNA ratio declined in the uterus, whereas it remained unaltered in the ovary. This decrease might be due to a significant decline in RNA concentration.

The DNA/Protein ratio was also significantly decreased in the ovary and uterus which could be related to the significant decline in protein levels. Thus it is likely that the process of transcription and translation would be affected in NaF treated mice. Further detailed studies on DNA repair mechanism on DNA polymerase activity as well as other enzymes involved in nucleic acid synthesis and metabolism are called for in the future.

In the present study, NaF treatment (60 days) induced complete loss of fertility and absence of implantation sites in NaF treated females when mated with control males. The loss of fertility might be due to irregularity in cyclicity related to altered hormone levels.

The present study thus elucidates that NaF brought about alterations in ovarian and uterine nucleic acid metabolism and had effects on reproduction. Since the structure and internal milieu of the uterus is maintained by priming of estrogen and action of progesterone, it is necessary to study the levels of these hormones and the ultrastructural changes in the ovary and uterus.

The present study also elucidates that supplementation of amino acids (glycine and/or glutamine) along with NaF manifested amelioration in all NaF induced effects, which was more pronounced with the combined administration.

Acknowledgement: The financial support provided by the Council of Scientific and Industrial Research (CSIR), New Delhi, to one of the authors (DP) is gratefully acknowledged.

REFERENCES

Tsutsui T, Suzuki N, Ohmori M. Sodium fluoride induced morphological and neoplastic transformation, chromosome aberrations, sister chromatid exchanges and unscheduled DNA synthesis in cultured Syrian Hamster embryo cells. Cancer Research 44 938-941 1984.
Sheth FJ, Multani AS, Chinoy NJ. Sister chromatid exchanges: A study in fluorotic individuals of North Gujarat. Fluoride 27 (4) 215-219 1994.
Singer L., Armstrong WD. Regulation of human plasma fluoride concentration. Journal of Applied Physiology 15 508-510 1960.
Guercio F, Cazzola D. The mechanism of action of several hypophyseal hormones: Influence of several poisons and of pancreatectomy on the gonadotropic and thyrotropic hormones. Archives of Science and Biology 27 405-413 1941.
Hanley TR, Calhoun LL, Kociba RJ, Grene JA. The effects of inhalation exposure to sulfuryl fluoride on foeta1 development in rats and rabbits. Fundamental and Applied Toxicology 13 79-86 1989.
Messer HJ, Armstrong WD Singer L. Influence of fluoride intake on reproduction in mice. Journal of Nutrition 103 1319-1326 1973.
Chinoy NJ, Walimbe AS, Vyas HA Mangala P. Transient and reversible fluoride toxicity in some soft tissues of female mice. Fluoride 27 (4) 205-214 1994.
Dhruva P, Milind VS, Narayana MV and Chinoy NJ. Effects of sodium fluoride on physiology of female mice and its reversal. Proceedings of the Academy of Environmental Biology 3 (2) 197-205 1994.
Chinoy NJ, Patel D. Ameliorative role of amino acids on fluoride-induced alterations in uterine carbohydrate metabolism in mice. Fluoride 29 (4) 217-226 1996.
Mejbaum W. Cited by Swift H. In: Chargoff E, Davidson JN (Eds). The Nucleic Acids Vol.2. Academic Press, New York 1955 pp 51-92.
Giles KW, Meyer A. An improved diphenylamine method for the estimation of DNA. Nature 206 93 1965.
WHO. A method for examination of the effect of plant extracts administered orally on the fertility of male rats. APF/IP, 99/4E, MB-50 1-12 1983.
Rubin JS, Joyner AL, Bernstein A, Witmore GF. Cellular response to DNA damage. In: Proceedings of the ULCA Symposium, Colorado, 1983. pp 615-624.
Strochkova LS, Zahvoronkov AA, Autsyn AP. Effects of fluoride on morphological modifications in Hela cell culture. Tsitologiya 26 299-306 1984.
Shashi A. Preliminary observation on alterations in Rabbit ovary DNA and RNA content in experimental fluorosis. Fluoride 27 (2) 76-80 1994.
Vorishilin SI, Plotko EG, Gatiyatulline EZ, Gileva EA. Cytogenetic effect of inorganic fluorine compounds on human and animal cells in vivo and in vitro. Genetica 9 422 1973.

FLUORIDE 31 (3) 1998, pp 143-148	International Society for Fluoride Research
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TABLE 1A. DNA levels (µ moles/100mg fresh tissue weight) in uterus and ovary of control and treated groups

	Uterus		Ovary
Groups	45 days	60 days	45 days	60 days

Control	886.34 ± 20.2	894.30 ± 18.1	1053 ± 36.3	1002.0 ± 21.2
NaF	484.79 ± 17.9^*	408.28 ± 16.6^*	658 ± 17.0^*	599.0 ± 18.2^*
NaF+Glycine	807.57 ± 12.2^*	907.72 ± 18.2^*	923 ± 16.3^*	976.0 ± 19.1^*
NaF + Glutamine	794.15 ± 19.9^*	865.28 ± 15.1^*	936 ± 19.9^*	941.44 ± 16.6^*
NaF+Glycine + Glutamine	907.12 ± 24.8^*	974.56 ± 14.2^*	1024 ± 32.6^*	1056.0 ± 21.9^*

Values are Mean + Std Error * P < 0.001

TABLE 1B. DNA ANOVA

Source of variation	SS	df	MSS	f(cal)	f(tab)	P value

Uterus Groups Residual	1104058.00 20732.00	8 18	138007.00 1151.00	119.80	3.71	<0.001
Ovary Groups Residual	849933.60 41481.00	8 18	106241.00 2304.00	46.10	3.71	<0.001

SS = Sum of squares df = Degrees of freedom MSS = Mean sum of squares

TABLE 2B. RNA ANOVA

Source of variation	SS	df	MSS	f(cal)	f(tab)	P value

Uterus
Groups	8280.65	8	1035.00	5.57	3.71	<0.001
Residual	3342.00	18	185.00
Ovary
Groups	42369.30	8	5296.00	17.00	3.71	<0.001
Residual	5604.60	18	311.00

SS = Sum of squares df = Degrees of freedom MSS = Mean sum of squares

TABLE 3B. DNA/RNA Ratio ANOVA

Source of variation	SS	df	MSS	f(cal)	f(tab)	P value

Uterus
Groups	62.55	8	7.82	7.28	3.26	<0.05
Residual	29.00	27	1.07
Ovary
Groups	16.07	8	2.01	4.17	3.71	<0.05
Residual	8.67	18	0.48

SS = Sum of squares df = Degrees of freedom MSS = Mean sum of squares

TABLE 4B. RNA/Protein Ratio ANOVA

Source of variation	SS	df	MSS	f(cal)	f(tab)	P value

Uterus
Groups	30.29	8	3.78	1.86	3.71	NS
Residual	36.47	18	2.02
Ovary
Groups	143.37	8	17.92	7.57	3.71	<0.001
Residual	42.57	18	2.36

SS = Sum of squares df = Degrees of freedom MSS = Mean sum of squares NS = Non significant

TABLE 5. Fertility rate (%) and implantation sitesof treated and control groups of mice

	No. of females mated/ No. of females pregnant		Fertility rate † † (%)		No. of implantation sites		Cyclicity
	45 days	60 days	45 days	60 days	45 days	60 days	45 days	60 days

Control	10/10	10/10	100	100	12.8± 0.9	12.5± 0.7	Regular	Regular
NaF	10/6	10/2	60	20	3.2± 0.5^**	1.5± 0.5^**	Irregular	Irregular
NaF+Glycine	10/9	10/9	90	90	8.5± 1.4^*	6.5± 0.3	Regular^†	Regular^†
NaF+Glutamine	10/9	10/9	90	90	8.0± 1.3^*	7.0± 1.1^*	Regular^†	Regular^†
NaF+Glycine + Glutamine	10/10	10/10	100	100	9.0± 1.5^**	9.0± 1.2^**	Regular^†	Regular^†

^†The oestrus cycle was found to be regular but duration of cycle was extended. ^† †Females:Males used in ratio of 2:1. ^*P< 0.001 ^P<0.01 At least 3 sets done for each group and mean values are presented.