Impact of nitrate intake in drinking water on the thyroid gland activity in male rat

Toxicology Letters 147 (2004) 27–33

Impact of nitrate intake in drinking water on the thyroid gland activity in male rat A. Zaki a , A. Ait Chaoui a , A. Talibi b , A.F. Derouiche c , T. Aboussaouira d , K. Zarrouck d , A. Chait e , T. Himmi a,∗ a

Laboratoire de Physiologie Animale, Faculté des Sciences et Techniques, Université Cadi Ayyad, Béni Mellal, Morocco b Service d’Endocrinologie, Hˆ opital Provincial, Béni Mellal, Morocco c Laboratoire de Recherche sur les Lipoprotéines, Faculté des Sciences Ben M’sik, Casablanca, Morocco d Laboratoire d’Anatomie Pathologique, Faculté de Médecine et de Pharmacie, Casablanca, Morocco e Laboratoire d’Ecophysiologie, Faculté des Sciences Semlalia, Marrakech, Morocco Received 13 May 2003; received in revised form 30 September 2003; accepted 2 October 2003

Abstract The purpose of this study was to determine the effects of nitrate on both the activity of the thyroid gland and other biological parameters. After 5-month treatment, nitrate 150 and 500 mg/l induced a significant decrease in the serum level of thyroid hormone T3. For T4, the 500 mg/l dose only reduced its plasma level. On the other hand, nitrate induced a dose-dependent increase in the weight of the thyroid gland. The histological study of the thyroid gland shows vacuolisation and an increase in the size of the follicles accompanied by a flatness of follicular epithelium with nitrate 150 and 500 mg/l. We concluded that the presence of high concentrations of nitrate in drinking water influence the growth, induce morpho-functional modifications of the thyroid gland and might be considered as a goitrigenic factor. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Tadla-Azilal; Nitrates; Rat; Thyroid gland; Thyroid hormones; Goitre

1. Introduction In man, previous studies have shown that a food iodine deficiency represents the principal factor of the development of the goitre (Aquaron et al., 1985; Iodice et al., 1992). This development is accompanied by an increase in the secretion of the pituitary hormone TSH and a decrease of plasmatic level of ∗ Corresponding author. Tel.: +212-23-48-51-12; fax: +212-23-48-52-01. E-mail address: [email protected] (T. Himmi).

the thyroid hormone T4 (Roux et al., 1983). The correction of this food deficit by supplementation in iodine induces a regression of the goitre (Roux et al., 1983). Recently, many studies showed that the goitre could have other origins. Indeed, in some patients, a goitre is observed despite a normal daily urinary iodine excretions (Rybakowa et al., 1992) or a correct daily iodine food supplementation (van Maanen et al., 1994). More precisely, some epidemiological studies in children showed that the goitre develops easily in zones where the contamination of drinking water by nitrates exceeds the permissible daily amount fixed by

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A. Zaki et al. / Toxicology Letters 147 (2004) 27–33

the WHO (1998), i.e. 50 mg/l in (Gatseva et al., 1998; Vladeva et al., 2000). In animals, goitrigenic effects of iodine deficiency or nitrate ingestion were also observed. Indeed, for rats, a food iodine deficiency induced the goitre (Stubner et al., 1987; Mooij et al., 1993). The correction of this food deficit by addition of iodine in the drinking water induces a regression of the goitre (Stubner et al., 1987). In the same species, the consumption of nitrates induced histological modifications of the thyroid gland (Horing et al., 1986; Gatseva et al., 1992 and 1996) as well as a decrease in the secretion of the thyroid hormones (Jahreis et al., 1991). Similar effects of nitrates on the thyroid function were observed in other animals (Jahreis et al., 1986, 1987; Georgiev et al., 1987; Zraly et al., 1997). In Morocco, no study concerning the effects of nitrates on the goitre development was made so far. In the Tadla-Azilal region, an agricultural area located in central Morocco, the water supply of the rural population is ensured primarily by the wells in which a nitrate pollution exceeding 150 mg/l is often observed. Many inhabitants in this area have a goitre in spite of the generalisation of the salt enriched by iodine in this area. The purpose of the present study is to show, in the rat, a possible effect of a treatment by nitrate on the weight, on the histological aspect of the thyroid gland, on the production of the thyroid hormones T3 and T4 and on the development of goitre. Nitrate being supplied by drinking water in amounts equivalent to those measured in certain sites of this area of Tadla-Azilal.

2. Materials and methods Sixty male Wistar rats weighing between 70 and 80 g were used and divided into five groups of 12 rats. Four groups received water containing respectively 50 (group I), 100 (group II), 150 (group III) and 500 mg/l (group IV) of potassium nitrate (Gatseva et al., 1996); the control group (group C) received tap water containing approximately 13.55 + 0.13 mg/l (S.D.) of nitrate. All rats were fed ad libitum with a standard food containing Iode ? nitrate 21.87+0.09 mg/kg measured by cadmium reduction method (Alary et al., 1980) and had free access to drinking water. In order to assess

the effect of nitrate on the growth, the body weight was determined monthly. The rats were killed by decapitation after a 5-month treatment. After sacrifice, the blood was collected in EDTA and immediately centrifuged at 3500 rpm, and the serum was aliquoted and stored at −20 ◦ C. The total proteins and the ureamia were measured by spectrophotometry, the total plasmatic concentrations of thyroid hormones; triiodothyronine (T3) and tetraiodothyronine (T4) were measured by radioimmunology (RIA-C GNOST® T3, international Cis bio). The thyroid glands were weighed, and then cut in fine sections 4 ␮m thick, coloured with Haematin-Eosin and observed by optical microscopy, using a graduated eyepiece in order to measure the epithelium thickness and the size of the follicles. The statistical study of the results used a variance analysis (ANOVA). The comparison of two averages was performed with t-test of Student-Fisher (significance level P < 0.05).

3. Results In our rats, the 5-month treatment with nitrate in the drinking water induced a significant and dose-dependent fall of the body weight gain (ANOVA, F = (5, 48), P < 0.001) (Fig. 1). This fall reached 16% in group III and 25% in group IV. Table 1 shows that all doses of nitrate, induced a significant and dose-dependent decrease in total protein plasma concentrations. This fall reached 15% in group I and 51% in group IV. Contrary to total protein levels, table shows that the nitrate induces a significant and dose-dependent inTable 1 Total proteins and urea serum levels of male rats after 5-month of treatment by nitrate in drinking water Total proteins (g/l) Group Group Group Group Group ∗ ∗∗

C I (50 mg/l) II (100 mg/l) III (150 mg/l) IV (500 mg/l)

P < 0.05. P < 0.01.

63.72 53.87 49 46.09 31.09

± ± ± ± ±

2.61 4.54∗ 1.41∗∗ 2.46∗∗ 3.23∗∗

Urea (g/l) 0.32 0.47 0.52 0.68 1.35

± ± ± ± ±

0.04 0.03∗ 0.02∗∗ 0.04∗∗ 0.05∗∗

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Fig. 1. Effect of nitrate in drinking water on the body weight of male rats during 5-month of treatment. ∗ P < 0.05,

crease in the plasmatic urea concentration. This rise reached 47% in group I and 322% in group IV. The analysis of the regression straight lines shows a positive correlation between the body weight and the total plasma proteins (r2 = 0.77, P < 0.01), and a negative correlation between total plasma proteins and the urea serum levels (r 2 = 0.65, P < 0.01). The histogram of thyroid gland weight of all animals including the reference group (Fig. 2) shows a significant difference between the control and the II–IV treated groups. Thus, the nitrate 50 mg/l was without effects on the weight of this gland (P > 0.05) but the nitrate at 100, 150 and 500 mg/l induced sig-

∗∗ P

< 0.01.

nificant dose-dependent increases in the weight of the thyroid gland. These increases reached 21, 45 and 77%, respectively. Table 2 shows the mean plasma concentrations of thyroid hormone T3 and T4. The effects of nitrate on these concentrations were studied in the control rats and in the groups III and IV. The chronic treatment by nitrate 150 mg/l reduced significantly the plasma levels of T3 by 34% (P < 0.05), but for T4, this fall reached 12% and was not statistically significant. The nitrate 500 mg/l reduced significantly (P < 0.05) the levels of T3 and T4 by 44 and 30%, respectively.

Thyroid gland weight (mg)

25 * *

20 * * *

15

10

5

0 Control

50 100 150 Nitrate concentrations (mg/l)

500

Fig. 2. Effect of nitrate in drinking water on the thyroid gland weight of male rats after 5-month of treatment. ∗ P < 0.05,

∗∗ P

< 0.01.

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Table 2 Plasmatic thyroid hormone levels (T3, T4) in male rats after 5-month of treatment by nitrate in drinking water

Group C Group III (150 mg/l) Group IV (500 mg/l) ∗

T3 (nmol/l)

T4 (nmol/l)

0.74 ± 0.12 0.49 ± 0.07∗ 0.41 ± 0.09∗

35.71 ± 3.06 31.57 ± 2.92 25.08 ± 1.08∗

P < 0.05.

The analysis of the regression straight lines shows a significant negative correlation between the weight of the thyroid gland and the plasma T3 level (r 2 = 0.31, P < 0.05) on the one side, and between the weight of the gland and the plasma T4 level (r2 = 0.37, P < 0.05) on the other side. The histological study of the thyroid gland shows that nitrate at 50 and 100 mg/l doses was without effects on the tissue structures of the gland. However in the groups III and IV we observed a vacuolisation and an increase in the colloidal volume of the follicles thyroid reached 14 and 21%, respectively. These modifications were accompanied by a flatness of the follicular epithelium which about 50% compared to the control (Figs. 3–5) (1500×).

4. Discussion Our results show that the consumption of nitrate induces in rats a dose-dependent reduction of the body weight gain. This conclusion is in agreement with that of other works (Fritsch et al., 1980; Jahreis et al., 1986; Ogur et al., 2000). This fall could be explained by a reduction of the water and food intake (Fritsch et al., 1980; Jahreis et al., 1991), or by an increase in the protein catabolism revealed by the low plasmatic level of total proteins and the high level of the uraemia observed in this study, or by a growth slowing down induced by the low plasma T3 and T4 levels. The long-term treatment by nitrate causes in our rats a dose-dependent increase of the thyroid weight similar to that observed following a daily food iodine deficit (Mooij et al., 1993), or following a high nitrate diet (Gatseva et al., 1999). This glandular hypertrophy is accompanied in our study, by a vacuolisation and a increase in the colloidal volume of the follicles and by a flatness of the follicular epithelium. Other morphological modifications of this gland were described by others following the consumption of nitrates in rat (Gatseva et al., 1996) and man (Gatseva et al., 1998). The histological disturbances observed in our study

Fig. 3. Micrography of the thyroid gland of the control rat colouring agent: Haematin-Eosin magnification (400×).

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Fig. 4. Micrography of the thyroid gland of the treated rat by nitrate 150 mg/l colouring agent: Haematin-Eosin magnification (400×).

are accompanied also by a reduction of T3 and T4 thyroid secretions The greater effect of nitrate one serum T3 than serum T4 could be due to the decrease of food intake that induced decrease in 5 -deiodinase activity resulting in a decreased peripheral production of T3 from T4. This effect seems to be due to a reduction in

the secretion of TSH also known for its morphogene action on the thyroide gland. In the rat, no study was carried out about the effect of nitrate on TSH production. However in man, an inverse relation ship was established between the volume of the thyroide gland and the plasmatic level of TSH (van Maanen, 1994).

Fig. 5. Micrography of the thyroid gland of the treated rat by nitrate 500 mg/l colouring agent: Haematin-Eosin magnification (400×).

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All these morpho-functional alterations of the thyroid gland after nitrate treatment are thus in favour of a goitrigenic effects of this substance. The mechanisms of action by which the nitrates alter the thyroid gland might be similar to those proposed in the literature. The fate of nitrates in the organism was the subject of many studies. Some works showed that the nitrates could be converted at the gastric level into nitrites, before their transformation into N-nitroso compounds known to be carcinogens for the stomach (Morales-Suarez-Varela et al., 1995). They can also be absorbed at the intestinal level, pass in the blood where they may induce methemoglobinemia (Gupta et al., 2000) or influence other functions such as the reproduction (Zraly et al., 1997; Panesar, 1999; Panesar and Chan, 2000) and the respiration (Gupta et al., 2000). The nitrates as well as nitrites seem to exert their effects in the organism after their reduction to nitric oxide (NO), as suggested by the following data. First, the use of an inhibitor of NO synthesis reduces the vascular expansion observed in the human thyroid gland during the goitre formation induced by a low iodine diet (Colin et al., 1995). Secondly, the expression of the enzyme of nitric oxide synthesis NOS III, in the human thyroid follicular cells and endothelial cells, suggests a possible role of NO in the functions of both cells (Colin et al., 1997). Thirdly, the long-term exposure to the NO donors (nitroprusside and S-nitrosoglutathione) inhibits significantly iodide transport and organification in cultured bovine thyroid cells, and reduces the differentiation of these cells (Costamagna et al., 1998). This possible inhibition of the iodine transport by NO would be exerted on Na(+)/I(−) symporter (NIS), an intrinsic membrane protein that mediates the active transport of iodide into the thyroid cell (De La Vieja et al., 2000). Finally, at the intestinal level, exogenic NO inhibits the spontaneous contractions of the jejunum of rabbit by activation of cGMP (Izzo et al., 1996), and in human thyrocytes, the nitroprusside stimulates cGMP production (Millatt et al., 1993). These results thus let us imagine that the nitrates introduced into the organism would exert their effects on the thyroid gland after their reduction in NO which activates cGMP. In conclusion, we suggest that the nitrate ingestion exceeding the standard level fixed by the WHO, i.e. 50 mg/l in drinking water, could be regarded as a factor

responsible for the genesis of the goitre in the area of Tadla-Azilal.

Acknowledgements We wish to thank Dr. BENOUHOUD M., Director of the Laboratory Anoual (Casablanca) for radio-immunological assays. We gratefully acknowledge Dr. El YAHYAI H. (Chief of Medical Centre of Analyse, Hospital Beni-Mellal) and Engineer BACHIRAT R. (Agence Hydraulique du bassin Oum Rbia) for their technical assistance and Dr. ORSINI J.C. for reading the manuscript.

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