Toxicology Letters, 57 (199 1) 309-3 18

@ 1991 Elsevier Science Publishers B.V. 0378-4274/91/S 3.50

ADONIS 037842749 IOOO84Y

TOXLET 02595

309

Effects of zinc toxicity on thyroid function and histology in broiler chicks

Carlton E. Dean’, Billy M. Hargis’ and Pamela S. HargiG

‘-Factdry of Nutrition, Departments of ‘~2Poulirv Science and 1 Veterinary Pathobiology, Texas Agricultural

Experiment Station, Texas A&M University Sjstern, College Station, TX (U.S.A.)

(Received 8 October 1990)

(Accepted 15 February 1991)

Kqv words: Zinc toxicosis; Thyroid function; Chickens

SUMMARY

Mechanisms of zinc (Zn) toxicity are incompletely understood and data regarding potential endocrine

alterations in Zn toxicity are scarce. To examine mechanisms of Zn toxicity, day-old chicks were pair-fed

diets containing 5280 ppm (Hz) or 73 ppm (CON) Zn. Impaired postnatal growth, independent of feed

consumption, and multiple endocrinopathies were observed following short-term (I-2 weeks) exposure

to the high Zn diet. Reduced levels of serum cholesterol, high-density lipoprotein cholesterol, and growth

hormone were associated with HZ feeding. Depressed levels of circulating thyroid hormones and histologi-

cal evidence that follicle area of thyroids from HZ birds was 63% less than CON indicated that impaired

growth of HZ birds may be caused, in part, by reduced thyroidal function.

INTRODUCTION

It is well established that both deficient and excessive levels of dietary zinc (Zn)

reduce growth and feed consumption in domestic and experimental animals [IA].

Most nutrition research regarding Zn requirements of man and animals has exam-

ined the consequences of deficiency on growth and development. Only recently has

attention been focused on potential consequences of excessive dietary Zn intake in

humans [5]. The various metabolic and growth-depressing effects of excess Zn in

many animals have been reviewed [6,7].

Zinc is regarded as relatively non-toxic to man and animals, particularly when ad-

Addressfor correspondence: Pamela S. Hargis, Faculty of Nutrition, Department of Poultry Science, Texas

Agricultural Experiment Station, Texas A&M University System, College Station, TX 77843-2472, U.S.A.

310

ministered orally. Most documented cases of Zn poisoning have involved acutely

toxic intakes of Zn in contaminated food, and pharmacological intakes resulting

from self-supplementation or use in treatment of various medical problems. Con-

sumption of food or drink following prolonged storage in galvanized containers has

also resulted in several cases of human Zn toxicity [8]. Symptoms of Zn toxicity in

response to an estimated intake of 225450 mg Zn include nausea, vomiting, epigast-

ric pain, abdominal cramps, and diarrhea [5]. Zinc intakes in the range of 100-300

mg/d have resulted in development of severe copper deficiency, alterations in immune

response, and disturbances in blood lipid profiles [9]. Several studies indicate that

moderately excessive Zn intakes (15-100 mg Zn/d) typically used with self-sup-

plementation can also have adverse metabolic consequences such as excessive copper

excretion [ 10, I l] and alterations in serum lipoprotein profiles [ 12,131.

Mechanisms of Zn toxicity are incompletely understood and data regarding poten-

tial endocrine alterations in Zn toxicity are scarce. Furthermore, no attempt has been

made to relate growth failure and other developmental effects of Zn toxicity to endo-

crine alterations. Recently, we observed impaired postnatal growth and altered

endocrine parameters in broiler chicks fed a toxic level of Zn. Here we report these

alterations in endocrine function with particular attention to morphological and

functional changes of the thyroid gland associated with high Zn intake.

MATERIALS AND METHODS

Experimental protocol

Two hundred day-old male chicks were weighed, wingbanded and randomly di-

vided into high Zn (HZ) and pair-fed control (CON) treatment groups (n= lOO/

group). Each group was randomly assigned to pens in starter batteries in 8 replicates

of approximately 12 birds/pen. HZ and CON birds were fed rations meeting Nation-

al Research Council recommendations for growing chickens [14]. Zn content of the

rations was calculated from National Research Council feed tables and Zn levels ad-

justed to 5000 and 50 ppm for the HZ and CON rations, respectively, by addition

of ZnO. Actual dietary Zn levels were determined by flame atomic absorption spec-

trophotometry on quadruplicate samples, which had been ashed overnight and dis-

solved in 0.3 N HCI. Aqueous working standards containing between 0 and 1.5 ppm

Zn were prepared from a stock standard solution (zinc reference standard, 1000 ppm,

Fisher Scientific Co., Fair Lawn, NJ) and utilized for calibration of the spectrophot-

ometer. Dissolved feed samples were further diluted in double-distilled deionized

water, and the zinc content of samples, determined from direct readings following

correction for dilution factors, was determined to be 5280 ppm for the HZ and 73

ppm for the CON treatment groups. HZ birds consumed feed ad libitum and CON

birds received only the amount of feed consumed per bird by the HZ birds on the

previous day. All birds received double-distilled deionized water ad libitum. Feed and

water were provided in stainless-steel pans to minimize contribution of Zn from

311

sources other than the feed. All birds were weighed weekly. Blood samples were ob- tained weekly from the wing vein of 10 randomly selected birds from each treatment group. Serum was separated by centrifugation and frozen at -20°C for further anal- ysis. Following 4 weeks of dietary treatment, thyroid glands and liver sample were excised from 5 birds/treatment. Thyroids were fixed in 10% buffered formalin for histological analysis. Liver samples were snap-frozen by immersion in liquid nitrogen and then maintained at -75°C in phosphate-buffered saline prior to determination of Td-5’-monodeiodinase activity [IS].

Serum analysis

Serum samples diluted 1: 1.5 in double-distilled deionized water were analyzed in duplicate for Zn content by atomic absorption spectrophotometry. Zn standards, prepared as described above, were utilized for calibration of the spectrophotometer. Total serum cholesterol was determined by an enzymatic procedure (No. 352-3, Sigma Chemical Co., St. Louis, MO). Serum high-density lipoprotein cholesterol (HDL-C) was determined following selective precipitation of very-low-density lipo- protein cholesterol and low-density lipoprotein cholesterol with subsequent assay of the supernatant for cholesterol (No. 352, Sigma Chemical Co., St. Louis, MO). Serum thyroxine (Td) and triiodothyronine (Ts) levels were determined by radioim- munoassay using a modification of the procedure of May [16] as described by Lien [ 171. Serum growth hormone (GH) levels were determined using a homologous radio- immunoassay recently validated in our laboratory [I 81.

Histology Histological sections of formalin-fixed, paraffin-embedded thyroid glands were

prepared and stained with Gill’s 3 hematoxylineosin (Fisher Scientific Co., Fair Lawn, NJ). Photomicrographs of a minimum of 2 randomly selected fields of each thyroid section were used for determination of mean follicle area by computerized morphometric analysis (Sigma Scan, Jandel Scientific, Corte Madera, CA).

Statistics

Data were analyzed using analysis of variance (General Linear Models, SAS Insti- tute, Raleigh, NC). Significance is reported as P< 0.05, except where noted.

RESULTS AND DISCUSSION

Toxicity of the level of Zn used in this study was strongly evidenced by the de- pressed body weights of HZ birds. Broiler chicks fed high levels of dietary Zn exhibit- ed significantly lower body weights as compared to CON despite equivalent feed in- take (Fig. 1). As might be expected, serum Zn levels of HZ birds were greater than CON at each sampling (Fig. 2). Reduced body weights and feed consumption have been observed in previous studies investigating effects of excessive Zn in both mam-

312

r 0 controt Lzs9 High Zinc

0 Control m High Zinc

O- Hatch 1

a

b hL 2

a

I b IL 3

a

1 b

AGE (week)

Fig. 1. Effect of high dietary zinc on body weight and feed consumption of male broilers. Parameter means

f SEM within ages differ (P < 0.05) if superscripts differ.

0 Control mS High Zinc

WEEK

Fig. 2. Effect of high dietary zinc on serum zinc levels of male broilers. Means f SEM within ages differ

(P < 0.05) if superscripts differ. Serum zinc was not determined at week 3 due to insufficient sample vol-

ume.

313

1uu

0 Control p;I9 High Zinc

2 WEEK

Fig, 3. Effect of high dietary zinc on serum growth hormone levels of male broilers. Means I SEM within

ages differ (P~0.05) if superscripts differ.

a 0 Control DY High Zinc

a

600

500

s

400 cn

5 P 300

200 l-llE!L b

100

0 I 1

0 Control 6Il High Zinc

i

WEEK

Fig. 4. Effect of high dietary zinc on serum thyroxine (T4) and triiodothyronine (Tr) levels of male broilers.

Parameter means + SEM within ages differ (P < 0.05) if superscripts differ. Serum Tj levels were not deter-

mined at week 3 due to insufficient sample volume.

314

malian and avian species [IA]. Several investigators have induced molting in laying

hens by administering high levels of dietary Zn [19-211. The effectiveness of Zn as

a molting agent has been hypothesized to be due, at least in part, to a marked reduc-

tion in feed consumption. Feed consumption usually decreases quickly with high Zn

intake followed by a reduction in growth. Low palatability of the diet may be partial-

ly responsible for the reduction in consumption of feed containing high levels of Zn

[22]. In the present study, feed consumption and growth were reduced significantly

after 1 week, but it is apparent from the feed consumption data (Fig. 1) that reduced

feed intake is not entirely responsible for the reduced growth of the HZ birds.

One possible mechanism of reduced growth, not related to reduced feed consump-

tion, is a Zn toxicosis involving an endocrinopathy. In order to investigate this possi-

bility, circulating levels of serum GH, T4 and Ts, as well as thyroid histology and

hepatic Td-S-monodeiodinase activity were evaluated. Serum GH levels of HZ birds

were significantly reduced as compared to CON values at weeks 2 and 3 (Fig. 3). Sim-

ilarly, serum T4 levels of HZ birds were significantly reduced from weeks 2 through

4 and serum T3 levels of HZ birds were diminished at weeks 1,2 and 4 (Fig. 4). These

data may indicate that high Zn intake altered production and/or secretion of the thy-

Fig. 5. Thyroid follicular cell hyperplasia and hypertrophy in a broiler chick fed a high zinc diet for 4 weeks

(A) as compared to a pair-fed control chick (B). Thyroid sections were sampled and prepared for histologi-

cal evaluation as described in ‘Materials and Methods’. x 212.

315

TABLE I

THYROID FOLLICLE AREA OF MALE BROILERS FOLLOWING 4 WEEKS OF HIGH DIE-

TARY ZINC

Follice area @mz)

Control High zinc

21321331 788k 183

Means k SEM significantly (P < 0.05) differed.

TABLE II

EFFECT OF HIGH DIETARY ZINC ON HEPATIC T,-S-MONODEIODINASE ACTIVITY OF

MALE BROILERS AT 4 WEEKS OF AGE

Enzyme activity (ng Ti/mg protein)

Control High zinc

3.0_+0.16 2.8kO.25

Means + SEM do not significantly (P> 0.05) differ.

roid hormones. In addition to alterations in thyroid function, the diminution in circu- lating GH levels may suggest either decreased anterior pituitary secretory function or decreased hepatic clearance. The possibility that the decreased thyroid function observed in the present study was due to diminished adenohypophyseal regulation of the thyroid was not evaluated in this study since the structure of avian thyroid- stimulating hormone (TSH) has not been determined and there is no radioimmuno- assay for determination of TSH in chickens. Regardless, a diminution in thyroid function is evident and is further supported by the histological observation that the thyroid tissue of HZ birds contained areas of follicular cell hyperplasia consisting of small follicles lined by high cuboidal epithelium. Such areas were much less pro- nounced in the thyroid tissue of CON birds (Fig. 5). Mean individual follicle area of thyroids from CON birds was greater than that of thyroids from birds fed 5280 ppm Zn (Table I). The observed diminution in circulating Ts was not due to dimin- ished extrathyroidal conversion. Following 4 weeks of dietary treatment, the activity of hepatic Tq-5’-monodeiodinase, the enzyme responsible for most extrathyroidal conversion of T4 to Ts, was not significantly altered (Table II). Thus, the combined findings of altered thyroid histology and depression in circulating thyroid hormones may indicate a hypothyroidism induced by Zn toxicity in the present study.

Inconsistent with this hypothesis of Zn-induced hypothyroidism is the observation that serum cholesterol and HDL-C levels were significantly and consistently de-

316

creased in the HZ birds as compared to CON (Fig. 6). Total serum cholesterol con-

centrations have generally been found to be elevated in human patients with hypo-

thyroidism and depressed in hyperthyroidism [23]. Consistent effects of high levels

of dietary Zn on serum cholesterol levels have not been reported [5]. However, several

investigators have observed a decrease in HDL-C levels in response to high dietary

Zn intake [12,13,24,2.5]. Therefore, it is possible that the reduced cholesterol and

HDL-C observed in the present study were due to metabolic effects not related to

thyroid function. Several studies indicate that high levels of dietary Zn increase cop-

per excretion [lo,1 1,261, and derangement in copper metabolism as a consequence

of Zn supplementation has been associated with decreased concentrations of HDL-C

[51. The results of this study indicate that the decreased growth observed in Zn toxicity

is not entirely related to diminished feed consumption. Furthermore, it is apparent

that multiple endocrinopathies are developed following a relatively short exposure

b

- 0 Control LSJ High Zinc

b

L 4

b

pL z

WEEK

0 Control LSY High Zinc

i

I 4

Fig. 6. Effect of high dietary zinc on total serum cholesterol and high-density lipoprotein-cholesterol

(HDL-C) levels of male broilers. Parameter means k SEM within ages differ (P <O.Ol) if superscripts

differ. HDL-C levels were not determined at week 3 due to insufficient sample volume.

317

(l-2 weeks) to elevated dietary Zn consumption in young growing chickens. The rela-

tionship of these endocrinopathies to the diminished growth observed following ele-

vated dietary Zn consumption remains to be established.

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