Growth inhibition induced by chronic dexamethasone treatment of foals

GROWTH INHIBITION INDUCED BY CHRONIC DEXAMETHASONE TREATMENT OF FOALS M. J. Glade PhD; L. Krook DVM; H. F. Schryver DVM, PhD, and H. F. Hintz PhD

SUMMARY

INTRODUCTION

Pony and horse foals were given daily intramuscular injections of dexamethasone, beginning at 6 months of age. Ponies were injected with 0 or 0.5 mgt 100 kg bodyweight for 3,8, or II months, or with 5.0 mgt 100 kg for II months. Horses were treated with 0 or 5.0 mgt 100 kg for 20 weeks. Rates of gain were inhibited (P<.OI) in both ponies and horses after 8 weeks of treatment with either dose. Weight loss occurred in ponies after 40 weeks oftreatment and in horse foals after 16 weeks, although feed intakes and dietary energy and nitrogen digestibilities were not effected by treatment (P>.IO). Urinary nitrogen loss increased within 3 months (P <.01), allowing little increase in total body protein during the period of normally rapid growth. Samples of left femur growth cartilage taken from ponies killed after 3 and 8 months were analyzed. Deoxyribonucleic acid content (ug/ mg dry cartilage) was doubled by treatment (P <.01), lactate dehydrogenase activities per cell and hexosamine and hexuronic acid contents were decreased 80-90% (P < .01), and hydroxyproline content decreased about 50% (P<.OI), compared to tissues from untreated ponies. Growth inhibition appeared to result from the production of abnormal growth cartilage, deficient in collagen and glycosaminoglycans. A metabolic shift from energy production to intra-chondrocyte energy storage, as reflected by decreased. lactate dehydrogenase activities, appeared responsible for the decreased production of cartilage components.

Glucocorticoid treatment of growing equines is not uncommon.F'" However, information describing the relationships of such treatment to rates of growth and weight gain is not available. While clinical treatment is only rarely continued beyond a few days, chronic treatment can be used as a model to study the extremes of glucocorticoid - induced effects. Subtle effects with potentially long-term consequences may thus be revealed. These studies were undertaken in an attempt to more clearly understand the interactions among prolonged glucocorticoid treatment, nutrition and growth of equines.

Author's address: Department of Animal Science, University of Maryland. College Park 20742. The bodyweight data appearing herein were previously presented at the 25th Annual Convention of the American Association of Equine Practitioners, Miami Beach, 1979. 198

MATERIALS AND METHODS Trial 1. Standardbred and thoroughbred foals (4 halfsiblings of each) were paired at weaning(6 months of age) by age and breed. One member of each pair received a daily intramuscular injection of dexamethasone a, 5 mg/100 kg bodyweight (BW), for 20 weeks. Foals were kept individually in 5 X 5 m. stalls and were allowed access to a dirt paddock with a water supply twice daily. They were fed a complete pelleted diet b at a rate of 2 kg/100 kg BW / day, which provided the required nutrients for foals." Bodyweight, heart girth and height at the withers were measured biweekly and drug dosages adjusted when necessary. Blood samples were drawn after 12 weeks and total plasma triglyceride concentrations were determined by a colorimetric method!

"Azium. Schering Corp.; Bloomfield, N.J. bChoice, Agway, Inc., Syracuse, N.Y.
Trial 2. At 6 months of age, 14 pony foals were assigned to the following treatments, 2., per group: daily intramuscular injections of either 0 or 0.5 mg dexamethasone/100 kg BW for 3, 8 or II months, or daily injection of 5 mg/100 kg BW for II months. Housing, feed and water were supplied as in Trial I. Blood samples were drawn after 0, 12 and 24 weeks and were examined to determine total plasma triglyceride concentration as described above. Ponies assigned to the long-term (II months) groups were subjected to metabolic balance studies after 2, 7 and 10 months. They were transferred to metabolic stalls which permitted the-1quantitative and separate collection of urine and feces. Collection periods began after 5 days of adjustment to the stalls and continued for 7 days, during which feed and water intake and fecal and urinary excretions were measured. Feed, fecal and urine contents of nitrogen were measured by macro-Kjeldahl.' The gross energy contents of feed and feces were determined by bomb calorimetry." Upon completion of their portions of the study (after 3, 8 or II months) ponies were sacrificed by barbiturate overdose. Thin mid-sagittal slices were cut through the right femoral proximal epiphysis with care taken to include a portion of the growth plate. Samples of growth plate cartilage were manually removed from the bone slices. Lactate dehydrogenase activities were immediately determined." Cartilage samples were then stored in liquid nitrogen and later thawed for determination of their deoxyribonucleic acid (DNA)5, hydroxyproline', hexosamine'? and hexuronic acid? contents. Either paired or unpaired Student's t-tests" or Gill's correlated univariate method for assessing treatment differences with repeated measurements? were applied to the data as appropriate. Metabolic balance and cartilage composition data were analyzed by two-way analyses of variance." Multiple comparisons among treatment levels or durations were made where appropriate using the least significant difference method."

RESULTS Appearance. Dexamethasone affected all treated animals, resulting in noticeably diminished stature, poor hair coat, lethargy, lameness and increasing reluctance to move after 8 weeks. Effects of treatment were less pronounced in the ponies than in the horses. Growth. In Trial I, the treated horses were smaller than controls in BW, height at the withers and heart girth circumference by week 12 (P< .05; Figure I). During the 20 weeks ofthe study, BW, withers height and heart girth of treated, foals increased only 13.2, 0.4 and 8.4%. respectively, compared to 49.0, 7.1 and 17.4% in the untreated foals (all P <.01). ' Because the pony foals were not closely related, they varied more than did the horses in initial BW. Therefore, their mean rates of BW gain over 8 week intervals are given in Table I, rather than their actual weights, While dOxygen Bomb Calorimeter; Parr Instrumcnt Co.• Moline, II..

November/December 1981

\lEIGHT KG

300 2~

200

240 220

200 HEIGHT AT WITHER$~

C"

~~I~~

no

HEARr

61RTHI CIt

155 150 145

140 135

no 10

12

14

16

18

20 WEEKS

Figure 1. Bodywelght (kg), height at the withers (em) and heart girth (em) ofhorsejoals (Mean±S.E.M.). 0·0 Control; .-. Treated with 5 mg dexamethasone per 100 kg bodywelght.

rates of gain decreased in all ponies with age, the treated ponies gained less weight throughout the study (P<.O I). In addition, the animals treated with 5 mg/100 kg BW initially tended to gain weight at the slowest rates and eventually lost weight more rapidly. Blood Analysis. Concentrations of plasma total triglycerides were elevated (P <.0 I) in the treated horses (100.7 ± 3.0 mg/ dl) at 12 weeks compared to the untreated horses (64.8 ± 1.8 mg/ dl). Concentrations of triglycerides were lower in the untreated ponies (P<.OI), averaging 20 tug] dl through 24 weeks, than in the ponies treated with 5 tng] 100 kg BW at 12 weeks (75.8 ± 1.4 mg/ dl) or in those treated with 0.5 mg/ 100 kg BW at 24 weeks (76.8 ± 2.0 tng] dl). Energy Digestibility. Neither age, treatment, nor the duration of treatment affected the digestibility of dietary energy (Table 2). Nitrogen Metabolism. The digestibility of nitrogen was not affected by treatment (Table 2). The urinary excretion of nitrogen was increased throughout treatment (P < .01), although the increase did not differ with treatment dose until 10 months. These increases resulted in decreased nitrogen retention by treated Table I Bodywelghts at a-week intervals (percent change from previous interval) in ponies (trial 2), Weeks 8 ........... 16.......•..• 24 ..•........ 32 .•.....•.•• 40 ...•...•... 46 ...•...•.••

Control

0.5mg A

35.70 29.83 18.08 12.18 3.48 2.33

18.30 14,07 7.41 7.26 -0.76 -1.19

5.0 mg A 16.83 B 4.I2 B 4.59 5.47 -2.99 -3.15

AJI,1g dexamethasone injected daily, per 100 kg body weight. "Means within a row with different underscore are different; P<.OI.

199

Table 2. En ergy digestibility and nitrogen metabolism in ponies. Level of Trealment A •8

Duration of Treatment 2 months 7 months 10 months

Gross energy, apparent absorpt ion (% of intake) 0.0 rng 83.31 E 83.58 76.40 N.S. c 0.5mg 81.42 83.16 76.45 N.S. 85.68 79.90 79.24 N.S. 5.0 rng Nitrogen. apparent absorption (% of intake) ' 0.0 mg 0.5 rng 5.0 rng 0.0 mg 0.5 mg 5.0 mg

78.50 77.50 78.50 76.39 74.77 74.50 78.09 82.50 82.00 Nitrogen, urinary excretion (% of intake) 30.95" 37.28 b 40.93 b

40.68< 61.52 d 62.46 d

57.95' 68.84 f 80.8 1g

N.S. N.S. N.S. P<.OID P<.OI P<.OI

AMg dex amethasone injected -daily, per 100 kg bodywieght. "Data a re means of 2 observations per group. cN.S. no significant differences within the row. I>Means within a row with different underscore area different. a.b.<.d·d.~Means within a column with different letters
=

animals throughout treatment (P < .01). The daily urinary loss of nitrogen increased in all animals with .a ge, although all animals remained in positive nitrogen balance throughout these studies. Cartilage Biochemistry. Growth plate cartilage specific activities of lactate dehydrogenase (LDH) and contents of deoxyribonucleic acid (DNA) and matrix components are given in Table 3. Specific activity ofLDH per cell was almost eliminated by 3 and 8 months of treatment (P <.01), while cartilage DNA content doubled (P < .01). Matrix contents (on a dry weight basis) of hexosamine, hexuronic acid and hydroxyproline were all decreased by dexamethasone treatment for 3 and 8 months (P< .01). Between 8 and 11 months, LDR specific activities per cell increased (P <.01) and DNA contents of cartilages decreased (P<.OI) in all groups. Hexosamine, hexuronic acid and hydroxyproline contents of cartilages from ponies treated with 5 mg/100 kg BW were not different . than those of cartilages from untreated animals. Hexosamine and hexuronic acid contents of cartilages from ponies treated with 0.5 mg/lOO kg BW did not change during this period, although hydroxyproline contents nearly doubled (P <.05).

DISCUSSION Decreased efficiency in the utilization of energy and nitrogen was evidenced in these studies by the decreased rates of gain in the dexamethasone treated horse and pony foals. This effect was not the result of depressed intakes or decreased energy or nitrogen digestibilities. An important factor contributing to the chronic inefficiency in energy metabolism was the shift in lipid metabolism from peripheral storage to increased mobilization. Energy sequestered in the blood of horses as very low density lipoproteins is only poorly utilized, 20 and may be accompanied by increased hepatic 200

Toble S, Lactate dehydrogenase specific activities and concentrations of matrix components in growth plate cartilages in pony foals.

Level of Treatment A •B

Duration of Treatment 3 months 8 months II months

Lactate dehydrogenase (spe cific activity unitsj ng DNA) 0.0 mg 0.5 mg 5.0 mg

3.50" O.64b

1.47< 0.12 d

19.53 g 28.68 24.03

P< .Olc . P< .01

Deo xyribonucleic acid (micrograms/ mg dry cartilage) O.Omg 0.5 rng 5.0 mg

5.45 E f 10.93

11.37· 24.74 d

--

3.14 1.44 2.30

P<.05 P< .01

Hexosamine (micrograrnsjrng dry cartilage) 0.0 mg 0.5 mg 5.0 mg

112.24' 21.46 b

132.05< 30.83 d

74.10< 25.50 f 60.36<

P<.05 N.S .I>

Hexuronic acid (rnicrcgramsj mg dry cartilage) O.Omg 0.5 mg 5.0 mg

116.11" 19.59 b

111.34< 26.90 d

68.47° 23.72 f 53.91"

P<.OI N.S.

Hydroxyproline (micrograrnsj mg dry cartilage) 0.0 mg 0.5 rng 5.0 mg

63.39 E 32.86 f

73.90· 49.81 d

80.20 86.37 85.12

N.S. P<.05

AMg dexamethasone injected daily, per 100 kg bodywieght. "Data are means of 2 ob servat ions per group. cMeans within a row with different underscore are different. I>lI!.S . = no significant differences with in the row . E.I·Means within a column with no sup erscripts are not significantly different, P<.05. a.bMeans within a column with different letters are different, P<.OI. CiMeans within a column with no superscripts are not significantly different, P<.05.

production of ketone bodies and the energy losses resulting from their excretion. II The increased urinary losses of nitrogen observed in these studies reflect the increase in protein catabolism induced by glucocorticoid treatment. 13 The energy demand of detoxifying the increased ammonia load may have played a role in wasting energy. Certainly the stimulation of protein degradation prevented the animals from enlarging their nitrogen stores. After 10 months of treatment daily urinary nitrogen losses nearly equalled the amount of dietary nitrogen absorbed, resulting in near balance, compared to the positive nitrogen retention of 9.3 g/ day observed in the growing untreated ponies. Decreased rates of bodyweight gain, eventual frank loss of weight and hyperlipemia are signs of malnutrition in the equine. 22.23 However, neither malnutrition nor malabsorption occurred during these studies. Instead, it appeared that the systemic shift to catabolism induced by dexamethasone treatment generated a condition of "metabolic malnutrition" by significantly diminishing the efficiency of nutrient utilization. The mechanism of growth inhibition also appeared to involve a shift in chondrocyte metabolism away from growth related activity. Lactate dehydrogenase activities have been shown to reflect the metabolic activity level of growth plate chondrocytes." Proximal left femur growth plate cartilage LDH activities per cell in treated animals EaUINE VETERINARY SCIENCE

were significantly reduced through 8 months of treatment. Similarly, cortisone treatment of rats has resulted in decreased chondrocyte LDH, phosphofructokinase and pyruvate kinase specific activities." Diversion of hexose metabolism from glycolysis to glycogenesis has been previously indicated by observations of a 70% increase in intrachondrocyte glycogen content in triamcinolone treated mice H through stimulation of glycogen synthetase activity." 27 Increased chondrocyte content of triglyceride, cholesterol and total lipids further supports the hypothesis that glucocorticoids enhance intrachondrocyte energy storage with concommitant failure to provide energy for synthetic pathways." The growth cartilage dry weight content of major matrix constituents (glycosaminoglycans and col1agen) were significantly reduced by dexamethasone treatment through 8 months, as indicated by the reduced contents of the components hexosamine, hexuronic. acid and hydroxyproline (Table 3). Deoxyribonucleic acid content was increased, indicating a greater cell: matrix ratio. These observations are consistent with previous reports I6,24,25 and suggest that matrix production as a whole was inhibited. Ratios of hexosamine, hexuronic acid and hydroxyproline to DNA in the growth plate cartilages from treated animals at 3 and 8 months were similar to the ratios in untreated animals at 11 months, supporting the hypothesis that dexamethasone treatment induced premature reductions in cellular synthetic activity, not unlike the "accelerated aging" of growth plate cartilage in glucocorticoid treated mice." Ratios of hexosamine and hexuronic acid to hydroxyproline were about 2:1 in untreated animals and less than 1:1 in ponies treated with 0.5 tug] 100 kg BW, through 8 months, indicating a greater inhibition of the synthesis -of chondroitan sulfate than of collagen.'? The lack of effect on the hexuronic acid :hexosamine ratios (which ranged between .84 and 1.03 and compare favorably to that of normal rat hyaline cartilage, 0.91 12) demonstrates that glycosaminoglycan production was inhibited before the incorporation of either hexose into the complex, but chondroitan that was produced was normal in mucopolysaccharide composition. However, subsequent sulfonation of chondroitan has been reported to be inhibited by glucocorticoids.t- v'v'! A common histologic observation of glucocorticoid treated animals has been the failure of metaphyseal capillaries to successful1y penetrate the transverse septa of the zone of provisional calcification, thereby effectively inhibiting longitudinal growth. Similar lesions were observed in these studies.'? The data presented above suggest the hypothesis that the organic matrix of the transverse septa, . being deficient in glycosaminoglycans and containing sulfa-deficient chondroitan moieties , either failed to present the proper "recognition signal" to the capillaries, or was resistant to the lytic enzymes released by those endothelial celIs. The increases in LDH activities per cell in all groups after II months may reflect the onset of sexual maturity. Enhanced maturation of stem cel1s would have November/December 1981

decreased their numbers and increased the overall average metabolic activity of the cartilage."

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