Abstract
Lead exposure is known to affect the pituitary-thyroid axis. Likewise, ascorbic acid (AA) has a protective action against lead poisoning. We examine the protective role of AA in lead-induced damage to the thyroid gland. The Wistar rats were divided into three groups: control that received 0.2% AA in drinking water throughout the experiment (15 days), intoxicated with lead acetate (20 mg/kg) intraperitoneally every 48 h for 15 days, and the experimental group treated with lead acetate and 0.2% AA in drinking water throughout the experiment. Plasma thyroid-stimulating hormone, triiodothyronine, thyroxine, and lead were determined. The thyroid gland was weighed, then epithelial cell height and nuclear volume were measured on histological slides. The results show that AA reduced the thyroid atrophy caused by lead acetate, as well as the loss of weight of the gland. In addition, it prevented the decrease of the hormone triiodothyronine, although the thyroxine hormone remained lower than the control values and the thyroid-stimulating hormone remains high. Our results indicated that AA could play a protective role in lead poisoning in the thyroid gland.
Keywords: lead-toxicity, thyroxin, TRH, triiodothyronine, TSH
Introduction
Lead (Pb) intoxication produces harmful effects on the hematopoietic, renal, hepatic, and gastrointestinal systems and is associated with various forms of cancer, nephrotoxicity, cardiovascular diseases, and central nervous system disorders [1]. Its degree of toxicity is related to the pathway of exposure, age, sex, level of intake, solubility, retention, duration of exposure, frequency of intake, rate and mechanisms of absorption, and level of excretion [2].
The exposure to Pb has been known to affect the pituitary-thyroid axis. The thyroid gland through its hormones, participates in a very important way in the metabolism of proteins, carbohydrates, and lipids. The secretion of thyroid hormones triiodothyronine (T3) and thyroxine (T4) by the thyroid depends on the stimulation of the pituitary thyroid-stimulating hormone (TSH), and this in turn of the hypothalamic thyrotropin-releasing hormone (TRH) [3]. The Pb intoxication increases TRH binding with rat anterior pituitary receptors [4]. Rats intoxicated with Pb show an increase in the epithelial height of follicular cells, as well as a reduction of the size of the thyroid gland follicles [5], stimulating the uptake of 131I by the thyroid gland [6] and alterations in its parenchyma [7]. However, contradictory reports are also available on the level of thyroid hormones after lead exposure. Serum T3 and T4 levels were not affected to lead in chronically exposed rats [8].
Nevertheless, another study has shown that lead administration in animals decreases serum T3 concentrations via reductions in hepatic type-I iodothyronine 5-monodeiodinase [9].
The protective action of ascorbic acid (AA) against Pb intoxication has been proposed. AA increases renal clearance and reduces the plasma concentration of Pb in rats [10–13]. Mice treated with Pb showed alterations in the frequencies of sister chromatid exchanges in bone [14] and micronuclei in bone marrow cell [15] and both effects were restored by the administration of AA. However, the protective effect of AA on the thyroid gland in lead intoxicated rats has not been studied.
Materials and Methods
Male Wistar rats (250 ± 25 g) were grown in our animal house from Universidad Autónoma de Aguascalientes. They were maintained in a controlled environment (light 14 h-dark 10 h and temperature 21°C) and were allowed access to food and water ad libitum. All procedures were in accordance with NIH Animal Care Guidelines and approved by the Bioethics Committee of the Autonomous University of Aguascalientes. Thirty animals were divided into three groups (10 rats per group): (i) Control AA: these animals received 0.2% of AA (Sigma-Aldrich, USA) in double-distilled drinking water throughout the experiment (15 days). (ii) Pb: rats were treated with lead acetate (20 mg/kg BW Sigma-Aldrich, USA) in 1 ml of double-distilled water intraperitoneally every 48 h for 15 days. The doses of lead acetate and the via of administration were similar to those reported by Thoreux-Manlay et al. [16] to induce endocrine alterations in rats. (iii) Pb + AA: the dosing schedule was the same as the Pb group, but the rats also received 0.2% of AA in double-distilled drinking water throughout the experiment. AA concentration was similar to that used by Dhir et al. [14] to obtain protective effects against Pb intoxication.
On day 15, all animals were sacrificed by exsanguination under deep anesthesia and the blood was collected for plasma determination of TSH, T3, T4, and Pb. Also, the thyroid glands were excised, weighed, and then placed in 10% buffered formalin and processed according to a conventional histological technique for hematoxylin–eosin staining. In the thyroid histological slides, the epithelial cell height and nuclear volume of 100 follicular cells per thyroid were measured. The size of the epithelial cell height was measured through an ocular micrometer calibrated with the stage micrometer. Two measurements of epithelial cell height per follicle were taken at each area between the apical and basolateral surfaces of follicular cells. The average size of the nuclear volume was calculated using the maximum diameter (D1) and the minimum diameter (D2) of nuclei considering the formulae Volume = ¾ (r1)(r2)(r3); where r1 = D1/2, r2 = D2/2 and r3 = (r1 + r2)/2 [17].
TSH concentration was measured by radioimmunoassay using the NIDDK rat TSH-Ria-1-9 Kit (National Pituitary Agency, Baltimore, MD, USA). The variation between the assay was 1.7% and the variation between assays was that of 5.4%. Commercial Kits were used to measure T3 and T4 by radioimmunoassay (Diagnostic Products Corp., Los Angeles, CA, USA. Catalogue number: T3: TKF31, T4:TKF41). The intra-and inter-assay coefficients of variation were 4.4% and 2.8 for T3 and 2.1 and 8% for T4, respectively. To the blood Pb level analysis, blood was digested in 5 ml of a nitric acid-perchloric acid solution at 110°C for 1 h. After filtering through a Whatman filter paper the digested blood was analyzed for Pb levels by using an atomic absorption spectrophotometer (Perkin Elmer Zeeman 5100, Norwalk, USA) and an HGA-600 graphite furnace. The absorption wave-length was 283.3 nm.
Data were analyzed by the use of ANOVA and differences between treatments were determined by the Tukey–Kramer multiple comparisons test. Values were expressed as means ± standard errors.
Results
The histological analysis shows that Pb modifies the structure of thyroid follicular epithelium. Also, treatment with AA recovers the morphology similar to that found in rats without Pb intoxication (Fig. 1) The treatment with AA reduced significantly the thyroid atrophy produced by Pb, as it avoided the decrease of epithelial height (7.0 ± 0.3 vs. 7.8 ± 0.3 μm) and increase of nuclear volume (8.2 ± 0.4 vs. 7.4 ± 0.3 μm3) of the thyroid follicular cells, showing similar values to control group without Pb (Fig. 2).
Figure 1.
Representative photomicrographs of histological sections of thyroid gland of rats intoxicated with lead acetate: (A) control thyroid (AA in drinking water), (B) thyroid of rats treated with Pb, and (C) thyroid of rats intoxicated with Pb and treated with AA. Sections were stained with hematoxylin–eosin. Magnifications were ×40. Arrows indicate follicular epithelium
Figure 2.
Epithelial height and nuclear volume of thyroid follicular cells. Rats intoxicated with lead acetate every 48 h for 15 days (Pb), treated with lead acetate with the same schedule dosing but also received AA throughout the experiment (Pb + AA) and controlled rats that received only AA solution for 15 days (Control AA). Data are expressed as mean ± SEM. *P < 0.05
The AA treatment also prevented the decrease of T3 plasma concentration (58.6 ± 2 vs. 62.2 ± 3.8 ng/dl), although the T4 remained significantly lower than control values (4.5 ± 0.6 vs. 6.6 ± 0.4 μg/dl) (Table 1). However, this AA effect did not avoid the TSH increase induced by Pb treatment (2.8 ± 0.1 vs. 2.2 ± 0.07 ng/ml) (Table 1). Atomic absorption spectrometry demonstrated that exposure increased the blood Pb level in intoxicated rats by the end of the experiment (202.4 ± 30.1 μg/dl), and AA reduced the increase in blood levels with statistical significance (132.6 ± 28.2 μg/dl; P < 0.02) whereas that in control group AA was 0.6 μg/dl. In all groups of rats exposed to lead, the blood concentration was statistically significantly higher compared with the control index (P < 0.01).
Table 1.
Plasma hormone levels of TSH, T3, and T4
| Control | Pb | Pb + AA | P value | |
|---|---|---|---|---|
| TSH (ng/ml) | 2.2 + 0.09 | 2.7 + 0.18 | 2.8 + 0.09 | P < 0.05 control vs. Pb, control vs. Pb + AA |
| T3 (ng/dl) | 60.3 + 0.36 | 47.0 + 0.27 | 59.0 + 0.18 | P < 0.01 control vs. Pb, Pb vs. Pb + AA |
| T4 (μg/dl) | 6.8 + 0.45 | 3.45 + 0.9 | 4.5 + 0.63 | P < 0.01 control vs. Pb, control vs. Pb + AA |
Rats treated with lead acetate every 48 h for 15 days (Pb), treated with lead acetate with the same schedule dosing but also received AA throughout the experiment (Pb + AA) and control rats that received only AA solution for 15 days (Control AA). Data are expressed as means ± SEM.
The correlation between the findings of thyroidal histology and hormone secretion showed that a reduction of epithelial height induced by Pb was accompanied by a decrease of hormone plasma levels of T3 and T4 and an increase of TSH as compared to controlled rats.
The weight of thyroid gland of rats treated with Pb was lower than that obtained from control rats (7.8 ± 0.3 vs. 10.1 ± 0.8 mg/100 g of BW P < 0.01). However, no significant differences were observed between the control group and those intoxicated with Pb and treated with AA (8.9 ± 0.2 mg/100 g of BW) (Fig. 3).
Figure 3.
Thyroid weight of rats intoxicated with Pb and treated with AA. Control group treated with AA, lead intoxicated with Pb Group (Pb), and lead intoxicated with Pb and treated with AA and Pb (AA + Pb). Values are expressed as mg/100 g of BW. NS = not significant and *P < 0.01
Discussion
The treatment with AA significantly reduced Pb effects on epithelial height, nuclear volume of thyroid follicular cells, and T3 plasmatic concentration. However, T4 remained without significant changes.
In our results, the rats intoxicated with Pb showed an increase in plasma TSH concentration, this fact may be due to a high affinity of the adenohypophyseal receptors to TRH and thus a secretion major [4]. Another possibility is that TSH release is less sensitive to feedback inhibition by thyroid hormone [18] or it could be both mechanisms. Similar results were obtained in rats intoxicated with cadmium, where plasma T3 and T4 concentrations were significantly decreased whereas plasma TSH failed to increase in response to low thyroid hormone levels [19].
The mechanism of action in lead poisoning rats could be through alterations in hypothalamic–pituitary–thyroid axis, specifically by disrupting of synthesis and secretion of the thyroid hormone. It is also possible that it may indirectly influence thyroid gland through inhibition of 5-deiodinase or by inducing hepatic microsomal enzymes, as T4-UDP-glucuronyltransferase. These mechanisms can reduce the circulating levels of T3 and T4 resulting in a release from negative feedback inhibition and increased secretion of TSH by the pituitary gland [9,20].
In this work, it was found that plasmatic concentration of T4 remained without significant changes. Lead influences thyroid hormone kinetics, however the pattern is inconclusive. Changes in total thyroid hormone levels might reflect the effect of lead on T4 binding proteins and not on the free thyroid hormone fractions.
The direct effect of Pb on thyroid glands is evident. The anisokaryosis observed in our study in follicular cells of animals intoxicated was similar to that obtained by Yagminas et al. [5] in nuclei of renal and hematic cells. However, they also observed an increase in epithelial height of thyroid follicles, while we do not observe differences in our study. This fact could be due to the chronic intoxication schedule used by them (91 days), versus subchronic intoxication (15 days) employed in our experiments.
It is well established that the atrophy of thyroid follicular epithelium produces primary hypothyroidism (low T3 and T4), which is in agreement with our results obtained in rats intoxicated by Pb; although Vyskocil et al. [8] did not observe changes in these hormones in rats with Pb for 3 and 4 months of treatment (chronic intoxication).
It has been described that Pb intoxication does not affect the weight of the thyroid gland in the rat [21]. However, in our results, a significant reduction in the thyroid weight was observed, which means atrophy of the gland and therefore a decrease in its function. This difference in results may be due to the intoxication scheme applied, where the dose and application time was lower.
In our study, both the weight of the thyroid gland and the recovery of the hormonal concentration in Pb + AA animals did not reach the same values as those of the control animals. This could be because the reduction in serum lead levels due to AA is partial and not total. Therefore, a longer period of administration of AA would be necessary.
To explain the protective effect of AA in lead intoxication, several mechanisms could be considered. One of them is through the increase of the renal excretion of Pb [10–13,22]. This fact can be supported since a reduction in blood Pb concentration was found at the end of the experiment. Another possibility is that Pb was sequestered in various tissues (a phenomenon associated with heavy metal exposure). It is also possible that the effect of AA may due from its ability to prevent the oxidative stress by trapping hydroxyl radical generated by exposure to Pb as it was demonstrated in rats intoxicated with ethanol, whom the exogen administration of AA significantly diminished hydroxyl radicals [23]. So that, the effects of AA may also have derived from the molecule’s antioxidant role against free radical-induced oxidative stress [13]. However, the dose used is important, since high concentrations of AA can have a pro-oxidative effect by reducing transition metals. Reduced transition metals react with hydrogen peroxide, leading to the formation of highly reactive and damaging hydroxyl radicals [24].
Conclusion
In conclusion, our results suggest a role for AA in the protection of the pituitary-thyroid axis after Pb intoxication. The involvement of multiple, interwoven, pathways may help to explain the beneficial effects of AA following Pb intoxication. Additional work will need to be done to help delineate the role and involvement of each pathway.
Conflicts of interest
There are no conflicts to declare.
Contributor Information
Denisse Calderón-Vallejo, Depto. de Morfología, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Avenida Universidad 940, C.P., 20131 Aguascalientes, Mexico; Depto. de Fisiología y Farmacología, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Avenida Universidad 940, C.P., 20131 Aguascalientes, Mexico.
María del Carmen Díaz-Galindo, Depto. de Fisiología y Farmacología, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Avenida Universidad 940, C.P., 20131 Aguascalientes, Mexico.
Andrés Quintanar-Stephano, Depto. de Fisiología y Farmacología, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Avenida Universidad 940, C.P., 20131 Aguascalientes, Mexico.
Carlos Olvera-Sandoval, Facultad de Medicina, Universidad Autónoma de Baja California, Avenida Alvaro Obregon s/n, Nueva, 21100 Mexicali, Mexico.
J Luis Quintanar, Depto. de Fisiología y Farmacología, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Avenida Universidad 940, C.P., 20131 Aguascalientes, Mexico.
Acknowledgements
We thank to AQB. Sonia Sofía Cruz Muñoz and Dra. María Consolación Martínez Saldaña for the technical support; and IBQ. Irma Hernández Jasso by the elaboration of the images.
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