The Effect of Humic Acid Substances on the Thyroid Function and Structure in Lead Poisoning

I Department of Physiology, Faculty of Medicine, Ordu University, Ordu, Turkey II Department of Nutrition and Dietetics, Faculty of Healthy Sciences, Artvin Coruh University, Artvin, Turkey III Department of Pathology, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey IV Clinical Biochemistry, Regional Research and Training Hospital, Erzurum, Turkey V Department of Animal Nutrition and Nutritional Disorders, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey VI ITekirdag Public Hospital, Tekirdag, Turkey VII Department of Biochemistry, Faculty of Veterinary Medicine, Ataturk University, Erzurum, Turkey.


INTRODUCTION
The thyroid gland mediates various metabolic processes in the body by secreting mainly thyroxine (T 4 ) and triiodothyronine (T 3 ) hormones.These hormones are derived from amino acids, and result from the iodination of thyroglobulin tyrosine residues.They are responsible for regulating the global metabolic activity of the body.The secretion of thyroid hormones is controlled by the thyroid-stimulating hormone (TSH) that is released from the anterior pituitary gland.Increased activity of the thyroid gland results in hyperthyroidism, whereas decreased activity results in hypothyroidism (Szkudlinski et al., 2002).Environmental, physiological, and genetic factors play a role in the development of both hyperthyroidism and hypothyroidism (Burger, 2004).The exposure to heavy metals is of the most important environmental factor that adversely affects the function of the thyroid gland (Cullent et al., 1984).
Lead (Pb) is one of the main heavy metals and non-essential toxic elements, and pose a risk to public health due to expanded urbanization and increased industrialization (Smith, 1984;Roper, 1991;Tong et al., 2000).It is widespread in nature and has been used in many industrial applications throughout history, such as manufacturing of batteries, some paints, glass, construction materials, agrochemicals, cosmetics, and fuel additives (Elwood et al., 1984).Once taken into the body through different routes, Pb accumulates in the organs and tissues.In the bloodstream, 85-90% of Pb is bound to erythrocytes, and the remaining 15-10% to plasma proteins (Lyn Patrick, 2006).Lead poisoning causes hematological, neurological, circulatory, and immunological pathologies, accompanied by biochemical changes, liver and kidney dysfunctions, and disrupt glucose metabolism (Al-Saleh, 1994;Lavicoli et al., 2003).Pb poisoning adversely affects endocrine glands, particularly the homeostasis of thyroid hormones, reproductive hormones and stress hormones (Zacharewski, 1998).
Chelating agents are used for the treatment of heavy metal poisoning.Humic acid substances (HAS) are water soluble, and include humic, fluvic, and ulmic acids derived from humus, breakdown product of organic substances in the soil (Islam et al., 2005).Previous studies have shown that humic acid regulates abnormal thyroid hormone secretion and act as immunomodulators (Laurberg et al., 2003).Due to their high molecular weight and functional groups (Schnitzer and Khan, 1972;Fan et al., 2004), it was hypothesized that supplementing diets with HAS reduces the adverse effect of heavy metals on the endocrine functions through forming chelates.This preliminary experiment was conducted to evaluate the prophylactic effect of supplementing HAS on hormonal and histopathological changes in the thyroid gland in laying hens exposed to Pb poisoning.

Animals, diets and experimental design
A total of 192 Lohmann White laying hens of 25 weeks of age randomly were assigned to one of the four dietary treatments.
A week before introducing the dietary treatments, all birds were fed the same iso-nitrogenous and isoenergy basal diet formulated to meet their nutritional requirements (NRC, 1994).Birds were then fed a basal diet, or the basal diet with 0.15% HAS (50.6% humic acid, 9.4% fulvic acid) (Farmagülatör XP, Farmavet International, Istanbul, Turkey), 0.3 g Pb/kg (lead acetate trihydrate, Acros Organics, New Jersey, USA), or the same levels of Pb+HAS.
Each treatment (n=48 birds) was replicated in 12 cages (42 x 48 cm), housing four hens each.The experiment lasted 10 days.During the adaptation and experimental periods, hens were subjected to a light regimen of 17L:7D.Feed and fresh water were provided ad libitum.

Blood sampling and biochemistry
At the end of the experiment, blood samples were collected from axillary veins of one randomly selected hen per cage (n=12 per treatment) into additive-free blood tubes.Blood samples were centrifuged at 1,500 g for 15 min, and sera were placed in Eppendorf tubes.The sera (250 µl) were first digested with a mixture of 2 mL HNO 3 (30%; Merck-KGaA, Darmstadt, Germany) and 3 mL H 2 O 2 (70%, Merck-KGaA) in a Microwave Digestion System (Berghoff, Eningen, Germany) for 25 min.The specimens were then subjected to analysis of elemental Pb using Inductively Coupled Plasma Emission Spectroscopy (Optima 2100 DV, ICP/ OES, Perkin-Elmer, Shelton, CT).Moreover, the sera were analyzed for T3, T4, and TSH levels using a diagnostic automatic analyzer (Modular Analytics Evo, F. Hoffmann-La Roche Ltd., Berlin, Germany).

Histopathology
After blood sampling, birds were sacrificed by cervical dislocation for harvesting the thyroid glands.Tissue samples were fixed in 10% buffered formaldehyde.Fixed tissues were embedded in paraffin blocks.The sections (5 µ) were stained with hematoxylin eosin (HxE), and evaluated under a light microscope after performing Masson's Trichrome and Periodic Acid Shift staining methods.

Statistics
Data were analyzed by one-way analysis of variance using the PROC GLM procedure (SAS, 2002).Statistical differences among group means were determined by the LSD option and considered significant at p≤0.05.Data were presented as least square mean ± standard error of the mean (LSM±SEM).

Thyroid Gland Histopathology
The thyroid tissues of the control hens did not show any histopathological changes (Figure 1).In the hens exposed to Pb poisoning, degenerative changes and necrotic areas were evident in the epithelial cells of the thyroid gland at various degrees of intensity (Figure 2A).Increases in connective tissue cell numbers in the interfollicular area and in the total amount of colloid with atrophic follicles were observed.Moreover, mononuclear cell infiltration, particularly lymphocytes in thyroid parenchyma and capsule and its surrounding fat tissue were evident (Figure 2B).Similar histopathological lesions were observed in the hens that were fed Pb+HAS, but at a lesser intensity.Follicular structure disappeared and colloid structure spreaded around to parenchymal and interfollicular areas in three cases (Figure 3A).In other cases, lesions were reduced and follicles presented normal histological appearance (Figure 3B).

DISCUSSION
Lead is one of the most harmful heavy metals and can accumulate in soft (i.e., liver, kidney) and hard (e.g., bone) tissues (Lyn Patrick, 2006;Gillis et al., 2012).Lead poisoning causes structural and functional changes in many organs, such as renal dysfunction, nervous system disorders, glucose metabolism abnormalities, liver dysfunctions, and hematological changes (Al-Saleh 1994;Lavicoli et al., 2003).In particular, it may cause anemia by reducing hemoglobin concentration in the hematologic system (Hilliard et al., 1973;Lynch et al., 1976).
Several agrochemicals are applied in crop production, and many poultry feedstuffs may be contaminated by environmental pollution.Lead can accumulate in the body, which may adversely affect animal health and pose a food safety risk.The Pb level of 0.3 g/kg diet was selected in the present experiment in order not to cause mortality, but to cause toxicity within 10 days (Vangris and Mare, 1974).Accordingly, mortality was not recorded, but the elevation of plasma Pb level was evident (Table 1).The changes in plasma Pb levels were associated with changes with the plasma levels of thyroid gland hormones (Table 1).
Lead adversely affects the production, secretion, and biological activities of thyroid and stress hormones and of hormone-related metabolism (Zacharewski, 1998).In the present study, Pb poisoning partially affected the thyroid hormones (Table 1), which may be related to the duration and level of Pb ingestion.Ibrahim et al. (2012) showed that lead acetate ingestion did not cause any significant reduction of plasma T 4 and T 3 levels.Similarly, in a study with 58 male gas station attendants and automobile mechanics with occupational exposures to high Pb levels, mean T 3 and T 4 levels did not differ (Singh et al., 2000).On the other hand, Robins et al. (1983) reported reduction of serum T 4 and FT 4 levels in humans exposed to excessive Pb levels.Dundar et al. (2006) evaluated the long-term and low-level Pb exposure among young individuals, and reported a negative correlation between blood lead and FT 4 levels, but no changes in TSH and FT 3 levels.In another study, high FT 3 level and low TSH level in the group of patients poisoned with lead compared with the control group were reported (Yılmaz et al., 2012).When Pb concentration exceeds 52 µg/dL, it triggers TSH secretion from the pituitary gland (Singh et al., 2000).In agreement with the present study, TSH level increased among workers exposed to lead (Gustatson et al., 1989;Lopez et al., 2000).
Lead can cause degenerative changes, satellitosis, neuronal vacuolation, neuronophagia, inflammatory changes and degenerative disorders in the nervous system, inflammatory cell infiltration and cytoplasmic vacuolation in the liver and kidneys (Taib et al., 2004;Ozsoy et al., 2011;Shalan et al., 2005).Degenerative changes, inflammatory cell infiltration, and interstitial connective tissue proliferation in the thyroid gland, indicating hypothyroidism, were observed in the present study (Figure 2).
Many agents, including vitamin C and L-carnitine, are applied for Pb poisoning treatment (Shaban El-Neweshy and Said El-Sayed, 2011;Flora et al., 2003).HAS are reported to alleviate abnormal thyroid hormone secretion and to act as immunomodulators (Laurberg et al., 2003).Nonetheless, HAS have multifunctional characteristics, such as to be able bind to metal ions to form chelates (Schnitzer and Khan, 1972).In the present study, dietary HAS supplementation partially alleviated feed intake and egg production reductions by 17.7% and 23.8%, respectively (data not shown) and reduced serum Pb level by 16.4% (Table 1) compared with the hens exposed to Pb poisoning and not supplemented.Moreover, it reduced serum TSH to normal levels (Table 1), which could be explained but its protective effects on the parenchymal cells of the thyroid gland.In addition to forming chelates with heavy metals, HAS has anti-inflammatory effects.The thyroid tissue showed similar, but less severe lesions when hens were supplemented with HAS (Figure 3).
In conclusion, in hens experimentally poisoned with Pb, FT 3 and FT 4 levels did not change, but TSH level increased by 167%.The increase in serum Pb and TSH levels of hens exposed to Pb poisoning was partially and completely normalized, respectively, by dietary HAS supplementation, and were accompanied by the

Figure 3 -
Figure 3 -Large colloid areas with lost follicular structure scattered in the parenchyma and interfollicular area (arrowhead) (H&E) (Panel A), and restoration of the thyroid gland to normal (arrowhead) (H&E) (Panel B) (Humic Acid Substances + Lead Group).

Effect of Humic Acid Substances on the Thyroid Function and Structure in Lead PoisoningTable 1 -
Table 1 summarizes serum Pb and thyroid hormone level results.The addition of Pb in the basal diet caused a 3.73-fold increase in serum Pb level compared with the control group (p<0.0001).HAS supplementation decreased serum Pb level by 16.5% compared with the Pb-poisoned group (p<0.05).Serum FT 4 level (0.71 ng/dL) and FT 3 :FT 4 ratio (5.49) were similar across the experimental groups.Lead poisoning caused a 2.67-fold increase in TSH level (p<0.05), and was suppressed by HAS supplementation to the control level.Effects of the dietary supplementation of humic acid substances (HAS) on serum lead and thyroid hormone levels of laying hens exposed to a 10-day lead (Pb) poisoning.
* Data are the least square mean ± standard error of the mean (SEM) (n=12 cages per group, 4 hens per cage).Different superscripts within the same rows differ (p<0.05).