Eur Arch Otorhinolaryngol (1990) 247: 267-270

European Archives of

Oto-RhinoLaryngology © Spnnger-Verlag 1990

Original investigations Hearing loss related to zinc deficiency in rats L. J. Hoeve I, J. Wensink 2, and I. R. A. M. Mertens zur Borg I lENT Department, Erasmus University, 3015 GD Rotterdam, The Netherlands 2Department of Radiochemistry, Interuniversity Reactor Institute, 2629 JB Delft. The Netherlands Received August 7, 1989 / Accepted August 11, 1989

Summary. Based on the observation that the zinc concentration in the cochlea of guinea pigs is very high, Shambaugh in North America has suggested that zinc plays an important role in hearing. Zinc deficiency is not infrequently present in growing children and elderly people, and thus might result in a hearing loss, which should be treated with zinc supplementation. The present experiment examined the effects of a zinc-deficient diet on hearing in 7-week-old rats. The auditory brainstem response threshold was measured in four rats before, during and after a zinc-deficient diet. Concentrations of zinc in the brain and in several other organs were measured, and the cochleas were examined microscopically. No hearing loss or morphological change in the cochlea of these animals was detected, although a clear zinc-deficient status was reached. Key words: Zinc deficiency - Hearing loss

Introduction Zinc deficiency is a well-established clinical entity that has been described by several authors [9, 11]. Children and older persons in particular may suffer from zinc deficiency due to an increased demand or insufficient nutrition. Based upon his observation that the zinc concentration in the cochlea of guinea pigs is very high. Shambaugh [13] suggested that sensorineural hearing loss might be a symptom of zinc deficiency. A zinc-deficient diet might lead to changes in cochlear function and thus produce a hearing loss. He examined patients with progressive sensorineural hearing loss and tinnitus (mostly presbycusis) for symptoms of zinc deficiency and measured serum zinc levels. If a zinc deficiency was found, he prescribed supplemental dietary zinc to these paOffprint requests to: L.J. Hoeve, ENT Department, Sophia Children's Hospital, Gordelweg 160, NL-3038 GE Rotterdam, The Netherlands

tients. He then reported some hearing improvement and diminution of tinnitus but in uncontrolled studies. In separate investigations Gersdorff et al. [3] also found a highly significant correlation between tinnitus and hypozincemia in humans. Zinc is a constituent of several important enzyme systems. These include carbonic anhydrase, carboxypeptidase, alkaline phosphatase, alcohol dehydrogenase, thymidine kinase, as well as D N A and R N A polymerase [11]. Zinc is needed for the growth and differentiation of cells and tissues, which might be due to its function in DNA and R N A formation [15]. It stimulates collagen production and the replication of fibroblasts in healing tissues. It also supports the thymus, thymocytes and granulocytes in their immune functions [11]. Zinc plays an important role in the structure and function of cell membranes of, for example, erythrocytes and cells of the skin, and in maintaining water balance [1]. In humans zinc deficiency has been associated with changes in the parotid gland [5], loss of taste and smell [12], and night blindness due to malfunction of the retinal rods [8]. Very severe zinc deficiencies have been found in children suffering from acrodermatitis enteropathica. This genetic disease is characterized by diarrhea, peripheral skin lesions, and infections. Untreated the disease may result in death. However, hearing loss has not been found in this condition [11]. Induced zinc deficiency in rats has resulted in varying disorders, depending on the timing of the diet [7, 8]. Intra-uterine zinc deficiency causes gross congenital malformations. In rat sucklings, growth retardation and abnormalities in brain development have been found to occur [11]. In older rats symptoms involve abnormal behavior without an abnormal brain structure [8], disturbances of feeding behavior, alopecia, dermatitis, impaired growth, poor wound healing, depressed gonadal function and impaired immunity [11, 14]. Hesse [6] reported altered neuronal transmission in the brain due to zinc deficiency in rats 32 days of age. This effect occurred in the hippocampal mossy fibers, where zinc concentration may be labile. However, this may be an

L. J. Hoeve et al.: Zinc-induced hearing loss

268 e x c e p t i o n a l s i t u a t i o n in t h e b r a i n , since o t h e r c e r e b r a l tissues d e m o n s t r a t e a stable zinc c o n c e n t r a t i o n , e v e n in n e a r - l e t h a l zinc deficiencies [7]. T h e b e h a v i o r o f t h e a u d i t o r y p a t h w a y in this r e s p e c t is n o t k n o w n , b u t can b e tested by brainstem response audiometry. S t i m u l a t e d b y S h a m b a u g h ' s o b s e r v a t i o n s in c h i l d r e n a n d o l d e r p a t i e n t s we i n v e s t i g a t e d t h e effects of a zincd e f i c i e n t diet o n the h e a r i n g o f rats after the critical p e r i o d for b r a i n d e v e l o p m e n t , thus c r e a t i n g a s i t u a t i o n c o m p a r a b l e to zinc deficiency in o t h e r w i s e h e a l t h y humans.

Materials and m e t h o d s

The study population consisted of eight male Wistar rats with normal hearing. The animals were 7 weeks old at the start of the experiment. They were pair-fed for 25 weeks, with four rats receiving a zinc-deficient diet (zinc content 0.6 + 0.2ppm), and the other four receiving supplementary zinc (50 ppm zinc as ZnSO4.7H20). Body weight was recorded once a week. Brainstem response audiometry was performed monthly to determine the binaural auditory brainstem response (ABR) threshold. Testing was performed while the animals were placed under general anesthesia (100 mg/kg ketamine i.m. and 10 mg/kg xylazine i.m.). Stimuli consisted of clicks with a duration of 100 gs and a repetition frequency of 10/s. The clicks came from a loudspeaker placed 10 cm from the animal's head. To determine the threshold, the intensity level was attenuated in steps of 10 or 5 dB near the threshold. The brainstem responses were recorded with subcutaneous needle electrodes placed at the tympanic bulla, the vertex and the back. The electrode signals were amplified x 105, filtered (band width 10-3000Hz, slope 24dB/oct.) and averaged 512 times. The ABR threshold was defined as the level at which peaks I and II were just discernible. Measurements of these rats with behaviorally normal hearing resulted in an average ABR threshold of 14dB SPL (116dB SPL-102 dB attenuation) (SD 2.9 dB, n = 42). Hearing loss was defined as a threshold shift of at least 10 dB. All rats were sacrificed at the end of the experiment. Zinc concentrations in plasma, bone, liver, muscle, kidney and brain were then determined after nitric acid/hydrogen peroxide wet digestion by atomic absorption spectrometry (Perkin Elmer Model H G A 76B). The soft tissues of the cochlea, particularly the organ of Corti and the vascular stria, were examined microscopically, using the method described by Hawkins and Johnsson [4]. Changes that can be observed with this technique include the disappearance of hair cells, pillar cells, nerve fibers and atrophy of cells in the vascular stria.

Results

O n e of t h e c o n t r o l a n i m a l s d i e d u n d e r a n e s t h e s i a d u r i n g an e a r l y r e c o r d i n g session, a n d its d a t a w e r e d r o p p e d f r o m t h e study. T h e o t h e r c o n t r o l a n i m a l s r e m a i n e d h e a l t h y , a n d d e m o n s t r a t e d a w e i g h t gain of a b o u t 6 0 % . I n c o n t r a s t , t h e w e i g h t gain for t h e g r o u p o f rats o n t h e z i n c - d e f i c i e n t d i e t was o n l y 21% ( T a b l e 1), a n d a l o p e c i a also o c c u r r e d . T h e zinc c o n c e n t r a t i o n s in t h e v a r i o u s tissues at t h e e n d of t h e e x p e r i m e n t a r e s h o w n in T a b l e 2. T h e c o n c e n t r a t i o n s in p l a s m a a n d b o n e w e r e significantly l o w e r in t h e zinc-deficient g r o u p t h a n in the c o n t r o l g r o u p ( S t u d e n t ' s t-test, P < 0.001). H o w e v e r , t h e con-

Table 1. Increase of weight (g) in zinc-deficient and control animals

Zinc-deficient animals

Control animals

Week

1

26

Increase

Week

1

26

Increase

Rat 1 Rat 2 Rat 3 Rat 4

193 173 172 188

237 196 209 237

23% 13% 22% 26%

Rat 1 Rat 2 Rat 3

181 195 183

295 322 289

63% 65% 58%

Average 182

220

21%

Average 186

302

63%

Table 2. Zinc concentrations after 25 weeks; mean values in gg/g (fresh weight) and standard deviations

Kidney Bone Brain Plasma Liver Muscle

Zinc deficient (n = 4)

Controls (n = 3)

14.5 65 11.7 0.21 20.2 12.0

17.7 176 11.8 1.08 21.2 10.4

+0.8 +7 +0.9 + 0.07 +3.7 +2.6

+ 1.5 + 25 + 0.1 + 0.07 + 1.2 _+ 1.0

Table 3. ABR threshold in zinc-deficient and control animals in dB SPL

Week 1

5

10

14

18

22

26

Zinc-deficient Rat i Rat 2 Rat 3 Rat 4 Average

10.5 13 14.7 13.8 13.0

9.2 5.5 10.5 9.2

18 13 20.5 17.2

6.3 12.2 12.2 5.5

13 11.3 13.8 13

8.8 8 12.2 11.3

8.8 11.3 16.3 11.3 11.9

Controls Rat 1 Rat 2 Rat 3 Average

13 20.5 13.8 15.8

15.5 14.7 15

14 19 18.8

12.2 13.9 10

14.7 10.5 15.5

10.5 11.3 10.5

13 14.7 18 15.2

Each figure represents the average of six measurements c e n t r a t i o n of zinc in the b r a i n tissue was t h e s a m e in b o t h groups. T h e A B R t h e s h o l d s m e a s u r e d in t h e course o f the exp e r i m e n t a r e given in T a b l e 3. T h e r e was n o significant c h a n g e in e i t h e r g r o u p d u r i n g t h e e x p e r i m e n t . I n the z i n c - d e f i c i e n t g r o u p , t h e a v e r a g e t h r e s h o l d was f o u n d at 1 3 . 0 d B S P L , with a s t a n d a r d d e v i a t i o n of 4 . 2 d B at the start of the experiment. At the end, the values were 11.9 a n d 4 . 4 d B , r e s p e c t i v e l y (24 m e a s u r e m e n t s ) . T h e values for t h e c o n t r o l g r o u p (18 m e a s u r e m e n t s ) a v e r a g e d 15.8 + 4.9 dB at t h e start o f t h e s t u d y a n d 15.2 + 4 . 6 d B at the e n d . M i c r o s c o p i c e x a m i n a t i o n r e v e a l e d no a b n o r m a l i t i e s in t h e c o c h l e a of e i t h e r g r o u p (Figs. 1 - 3 ) . T h e h a i r cell c o u n t was n o r m a l in b o t h g r o u p s , t h e r e w e r e n o missing p i l l a r cells, a n d t h e v a s c u l a r stria a p p e a r e d n o r m a l .

L. J. Hoeve et al.: Zinc-induced hearing loss

269

Fig. 1. The organ of Corti of a control rat. × 420

Fig. 2. The organ of Corti of a zincdeficient rat showing normal histomorphology. × 420

Fig. 3. The vascular stria in the cochlea of a zinc-deficient rat showing normal structures. × 420

270

L.J. Hoeve et al.: Zinc-induced hearing toss

Discussion

References

As the zinc-deficient diet was not started until the rats were 7 weeks old, we did not expect gross malformations or anatomical disorders in the brain to develop. However, we anticipated that functional changes in the normally developed cochlea and auditory pathway were more likely to occur. Between the age of 7 and 32 weeks, the normal weight gain of healthy rats with free access to food amounts to approximately 200%. The reduced weight gain of the control animals (60%) in the present study was due to the limited amount of food provided. As for the study group (21% weight gain), growth retardation coupled with alopecia is a well-known sign of zinc deficiency (Table 1). The low zinc concentrations in the plasma and bone of our animals confirmed this diagnosis. In the present experiment the zinc level in the brain of the rats did not change. This finding is in agreement with those studies reported by Kasarksis [7]. According to Shambaugh [13] the zinc concentration in the cochlea of guinea pigs does not change with zinc deficiency either. Still, these observations do not support his theory that hearing loss can result from zinc deficiency. In our experiments the rats with a zinc-deficient diet demonstrated a convincing zinc-deficient status. In our present study the hearing thresholds measured indicated that all rats had normal hearing [2, 10], and that the hearing levels did not change more than 10 dB during the experiment. Microscopic examination of the cochlea did not reveal changes in the organ of Corti or in the vascular stria. These findings show that zinc deficiency in young but mature rats does not result in hearing loss, nor does it produce microscopic changes in the structure of the cochlea. Further, these results do not support Shambaugh's suggestion that zinc deficiency might lead to hearing loss in children and the elderly.

1. Bettger WJ, O'Dell BL (1981) Minireview. A critical physiological role of zinc in the structure and function of biomembranes. Life Sci 28 : 1425-1438 2. Fay FF (1988) Hearing in vertebrates: a psychophysics databook. Hill-Fay, Winnetka, Ill., p 363 3. Gersdorff M, Robillard T, Stein F, Declaye X, Vanderbemden S (1987) A clinical correlation between hypozincemia and tinnitus. Arch Otorhinolaryngol 244 : 190-193 4. Hawkins JF, Johnsson LG (1976) Microdissection and surface preparations of the inner ear. In: Smith CA, Vernon JA (eds) Handbook of auditory and vestibular research methods. Thomas, Springfield, Ill., pp 5-52 5. Henkin RI (1978) Zinc, saliva and taste: interrelationships of gustin, nerve growth factor, saliva and zinc. In: Hambidge KM, Nichols BL (eds) Zinc and copper in clinical medicine. SP Medical and Scientific Books, New York, pp 35-48 6. Hesse GW (1979) Chronic zinc deficiency alters neuronal function of hippocampal mossy fibers. Science 205:1005-1007 7. Kasarksis EJ (1984) Zinc metabolism in normal and zinc deftcient rat brain. Exp Neurol 85 : 114-127 8. Pfeiffer CC, Braverman ER (1982) Zinc, the brain and behavior. Biol Psychol 17 : 513-532 9. Prasad AS (1976) Deficiency of zinc in man and its toxicity. In: Prasad AS, Oberleas D (eds) Trace elements in human health and disease, vol I. The nutrition foundation. Academic Press, New York, pp 1-20 10. Pujol R, Carlier E, Lenoir M (1980) The sensory hair cell. Ontogenetic approach to inner and outer hair cell function. Hear Res 2: 423-430 11. Sandstead HH, Evans GW (1984) Zinc. In: Olsen RE (ed) Present knowledge in nutrition. Nutrition Foundation, Washington, DC, pp 479-505 12. Sandstead HH, Vo-Khactu KP, Solomons N (1976) Conditioned zinc deficiencies. In: Prasad AS, Oberleas D (eds) Trace elements in human health and disease, vol I. The nutrition foundation. Academic Press, New York, pp 33-49 13. Shambaugh GE (1985) Zinc and presbycusis. Am J Otol 6: 116-117 14. Underwood EJ (1977) Zinc. In: Underwood EJ (ed) Trace elements in human and animal nutrition. Academic Press, New York, pp 196-242 15. Vallee BL (1983) A role for zinc in gene expression. J Inherited Metab Dis 6 [Suppl I] : 31-33

Acknowledgements. The study was supported by the Heinsius Houbolt foundation. Assistance in the completion of the study was provided by B. G. S. Groen, B. de Vrijer, and A. A. Polak.

Hearing loss related to zinc deficiency in rats.

Based on the observation that the zinc concentration in the cochlea of guinea pigs is very high, Shambaugh in North America has suggested that zinc pl...
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