Dermal Chemical Response to Analogues of Dilantin* J

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DERMAL CHEMICAL RESPONSE TO ANALOGUES OF DILANTIN* J. C. bUCK, Pn.D.

Dilantin sodium is known to be able to producetral soluble and acid soluble collagen respec- gingival hyperplasia (1) as well as to increase thetively (5). Both the extracts and the whole skin samples dermal concentration of insoluble collagen andwere hydrolyzed in 4K HC1 at 1000 C. for 8 hours. scleroprotein (2). Shafer (3) has indicated thatThese hydrolysates were analysed in triplicate for while most of the chemical analogues of Dilantintheir nitrogen (6), hexosamine (7) and hydroxy- were not capable of increasing the rate of fibro-proline (8) content as a measure of their protein, mucopolysaceharides and glycoproteins (9) and blast proliferation in tissue culture, additions ofcollagen (10) content. The difference between the either Dilantin, 1-allyl ,5phenyl hydantoinatetotal dermal concentration of these materials and or 5-methyl, 5 phenyl hydantoinate, into the in-the sum of their soluble concentrations was cal- cubation media of fibroblasts did result in almostculated. The insoluble dermal concentration of identical degrees of stimulation of cell growth.each material was therefore obtained. The results were expressed as the mean of replicate analyses We have assayed the effects of these Dilantinand their standard deviations for each group of 6 analogues upon the dermal chemistry of rats,animals in gzmoles of hexosamine or hydroxypro- and compared the results with those obtained line or in mmoles of nitrogen per g of fresh, cleaned from both normal and stressed control rats. Therat skin. The standard deviations of either groups of individual animals (totals) or of replicate analy- results are presented below. ses of duplicate extracts prepared were never more than 8% of the mean. Means differing by more MATERIALs AND METHODS than two standard deviations were significantly different (p < .05). Forty-eight male Sprague-Dawley rats (250—280 g) were divided into eight groups of six animals each, and six of these groups were subjected to 10 EXPERIMENTAL AND RESULTS daily intraperitoneal injections of 1.0 ml of isotonic saline containing 25mg of one nf the fol- The results of the analysis of whole rat skin lowing compounds in suspension: Dilantin,after 10 daily intraperitoneal doses of the various sodium; 1-propyl-5 phenyl hydantoin(I);materials described above are presented in Table 5-methyl-S phenyl hydantoin (II); 3-methyl-5, 5I. This data indicates that only with the admin- diphenyl hydantoin (III); 1-allyl-l-S phenylistration of cmpd. IV was there a marked in- hydantoin (IV); 1, 5-dimethyl-5 phenyl hydantoin (V). One group of animals was subjected to 10crease in dermal hydroxyproline concentration daily injections of isotonic saline alone, as a con-equivalent to that produced by Dilantin, while trol of the stress of such injections (4). The finalthe stress control animals demonstrated a sig- group of rats was retained as controls. After 10nificant decrease in dermal hydroxyproline con- days, all animals were sacrificed and 2g samples of abdominal skin were removed, shaven and dis-centration. The total hexosamine concentration sected clean of adhering fat, fascia and muscle.of the skin was essentially the same with all the These tissues were then minced and O.5g aliquotsdrugs studied, but only Dilantin administration removed. Other portions of the tissues were lyo-resulted in a profound increase in dermal ni- philized for the gravimetric determination oftrogen concentration. Compounds I, IV and V water. The remaining tissues from each group of 6 rats were pooled, and duplicate 4g samples ofall resulted in concentrations of total dermal pooled tissue were extracted sequentially in thehydroxyproline and nitrogen which were sig- cold with 0.15M NaCl, 0.SM NaCl and 0.SMnificantly above those found in the control ani- citrate buffer (pH 3.6) as has been described pre-mals, however. viously (2, 5). These extractions have been shown Collagen is known to contain about lpmole to remove quantitatively ground substance, neu- of hydroxyproline and about 13.3Mmoles of ni- Supported in part by grant A-3899 from thetrogen per mg of protein (11). From the data of N.I.A.M.D., P.b.S., Bethesda 14, Maryland. Table I, the concentration of dermal collagen per Received for publication April 10, 1962. *Fromthe Biochemical Research Laboratory,gm of fresh skin and the amount of collagenous Children's Hospital Research Foundation, Wash-nitrogen may be obtained. The difference be- ington 9, D. C. and Department of Biochemistry,tween this latter value and the total dermal Georgetown University Medical Center, Wash- ington 7, D. C. concentration of nitrogen is therefore due largely 89 90 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY to non-collagenous protein, since the concentra-II, and show that none of the compounds used tion of non-protein nitrogen has been shown tocould increase the dermal protein concentration be quite small in the tissue (5). Assuming thator decrease the water and fat concentration to non-collagenous dermal proteins contain 16%the same degree as did Dilantin. Only compounds nitrogen (11), then the concentration of theseII and III resulted in a significant increase of proteins may also be obtained from the data ofprotein concentration above the control or nor- Table I and expressed in terms of percent tissuemal concentration, while none of the compounds weight. Lyophilization of the various tissues per-resulted in any significant changes in dermal mitted the water content of the dermis to beweight or fat. determined. The difference between the sum of None of the compounds resulted in an increase the collagen, protein and water content in per-above control values in the concentration of cent total weight of the skin and 100% must beisotonic saline soluble hexosamine, or nitrogen due to fat. These results are presented in Tableas shown in Table III, except Dilantin. There- fore this ability of Dilantin to increase the con- TABLE I centration of "ground substance" appears to be Analysis of whole rat skin after the administrationan unique property of this compound. of 10 daily doses of various The concentration of neutral soluble collagen Dilantin analogues in rat skin was significantly increased above control values by all of the drugs studied, as shown Compound Hypro Heat Nt in Table IV, although this increase was quan- pmoles/g jtmoles/g snmsles/g titatively less with Dilantin administration than Control 190 107.5 .4 4.3 .2' with the other compounds studied. Similarly Dilantin 310 158.4 .5 8.1 .5 the amount of hexosamine found in the 0.51W Cmpd. I 226 12*8.5 .5 5.0 3* NaC1 fraction was increased above the control Cmpd. II 193 12*8.5 .55.0 3*with drug administration. This increase was es- Cmpd. III 179 10*7.2 4*47 3* sentially similar for all drugs studied. Cmpd. IV 280 158.7 .45.5 3k Citrate soluble collagen concentration, how- Cmpd. V 230 14*8.4 .5 4.9 3*ever, was decreased below the value of the con- Normal 240 15*7.7 .44.9 3* trols by Dilantin, whereas all the other deriva- * Significantlydifferent from Dilantin (p < .05).tives studied resulted in significant increases in tHypro =hydroxyproline;Hex =hexosamine; the dermal concentration of this material, as N =nitrogen. shown in Table V. Again, only Dilantin admin- Italicized means are significantly differentistration resulted in significant amounts of hexos- from control (p < .05). amine becoming soluble in this solvent.

TABLE II The composition, in per cent fresh weight, of cleaned rat skin from animals after the administration of 10 daily doses of Dilantin analogues

% Composition Compound Collagen Protein water Fat

Control 19.0 1.0* 15.0 1.5* 55 5 11 Dilantin 31.0 1.5 36.0 3.7 30 3 3 Cmpd. I 22.6 1.2* 17.4 1.8* 50 4* 10 Cmpd. II 19.3 1.2* 20.7 2.0* 50 5* 10 Cmpd. III 17.9 1.0* 28.1 2.7* 45 4* 9 Cmpd. IV 28.0 1.5 16.0 1.5* 46 5* 10 Cmpd. V 23.0 1.4* 17.0 1.5* 50 5* 10 Normal 24.0 1.5* 15.0 1.5* 50 4* 11 * Significantlydifferent from Dilantin (p < .05) Italicizedmeans significantly different from the control (p < .05) CHEMICAL RESPONSE TO DILANTIN ANALOGIJES 91

The difference between the sum of the solubleof this drug. Only compound IV resulted in a materials described above, and the total dermalsignificant increase in insoluble collagen concen- concentration of each component permits thetration above the control value, however, while calculation of the concentration of insoluble hy-compounds II and IV did result in significant droxyproline, hexosamine and nitrogen to beincreases above the control values of insoluble made. The effect of the various hydantoinatesdermal nitrogen. and the trauma of injection upon the concentra- The concentration of insoluble non-collage- tions of these insoluble materials is shown innous protein (scleroprotein) in these tissues was Table VI. Dilantin administration resulted incalculated as described previously from the data profound increases in the amounts of insolubleof Table VI, as were the ratios of collagen and collagen and nitrogen; increases that were nothexosamine to scieroprotein. These results, pre- quantitatively duplicated by the five derivativessented in Table VII, indicate that while there was an increased amount of scleroprotein found TABLE III with the trauma of injection, the administration Analysis of the 0.15 M NaCI extract of rat skin after of either Dilantin or compounds I, II, III and the administration of 10 daily doses of IV resulted in increases in scleroprotein signifi- various Dilantin analogues cantly larger than that of the control. The degree of increase in scleroprotein concentration pro- Compound Hyprof Hexf Nt duced by Dilantin was not equaled by the other jmo1es/g zmoles/g mmoles/g drugs, however. Again, Dilantin was unique in Control 3.6 .2*3.2 .2*0.63 .03* its ability to increase the ratio of insoluble hexos- Dilantin 6.0 .44.3 .3 0.80 .04 amine to scleroprotein above that of the control. Cmpd. I 4.2 •3*2.7 .2*0.56 .03* Cmpd. II 4.2 •3*2.0 .2*0.62 .03* DISCUSSION Cmpd. III 1.8 .2*2.4 .2*0.51 .02* Cmpd. IV 4.2 •3*2.2 .2*0.47 .02* The dermal chemical response to Dilantin ad- Cmpd. V 4.8 3*2.6 .2*0.56 .03* ministration has been shown (2, 11) to involve Normal 4.7 3*3.4 .2*0.58 .04 changes in four major components of rat skin: 1) an increase in total and insoluble collagen; * Significantlydifferent from Dilantin (p < 2) an increase in insoluble non-collagenous pro- .05). tein; 3) a decrease in dermal fat (12) and, 4) a t Hypro hydroxyproline; Hex =hexosamine; N=nitrogen. decrease in dermal water. Despite the ability of Italicized means are significantly different compounds II and IV to increase the rate of fromcontrol (p < .05). fibroblast proliferation (3), amounts of these

TABLE IV Analysis of the 0.50 M NaC1 extract of rat skin after the administration of 10 daily doses of various Dilantin analogues

Compound Hyprof Hext Nt

j.nwles/g ,.mo1e/g mmoles/g Control 8.6 •4* 1.4 .1* 0.41 .02* Dilantin 13.4 .7 2.3 .1 0.63 .03 Cmpd. I 23.4 1.8* 2.2 .1 0.56 .04 Cmpd. II 17.4 •7* 1.8 .1* 0.41 .02* Cmpd. III 22.2 1.6* 1.8 .1* 0.51 .02* Cmpd. IV 21.0 1.0* 2.6 .1 0.56 •Ø3* Cmpd. V 29.0 .2* 2.2 .1 0.83 .04* Normal 20.4 1.5* 1.6 .1* 0.70 .04

* Significantlydifferent from Dilantin (p < .05). fHypro =hydroxyproline;Hex =hexosamine;N =nitrogen. Italicizedmeans are significantly different from control (p < .05). 92 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

TABLE V Analysis of the 0.5M citrate extract of rat skin after the administration of 10 daily doses of various Dilantin, analogues

Compound Hyprot Hext Nt

zmoles/g mo1es/g mmoles/g Control 27 1* 0* 0.82 .04* Dilantin 19 1 0.43 .04 0.98 .05 Cmpd. I 42 2* 0* 0.93 .05 Cmpd. II 38 2* 0* 0.81 •Q4* Cmpd. III 43 2* 0* 0.72 .04* Cmpd. IV 48 2* 0* 0.81 .04* Cmpd. V 61 3* 0* 1.32 .07* Normal 8.0 .04* 0* 0.48 .02* * Significantlydifferent from Dilantin (p < .05). fHypro=hydroxyproline;Hex =hexosamine;N =nitrogen. Italicized means are significantly different from control (p < .05).

TABLE VI TABLE VII Analysis of the insoluble fractions of rat skin after Non-collagenous sciero protein and insoluble 10 daily doses of varions hexosamine and hydroxyproline in rat skin Dilantin analogues after the administration of 10 daily doses of Dilantin analogues Compound Hyprof Hexf Nf Compound Scieroprotein Hyprot/mg. Hext/mg. pmoles/g ,moles/g mmoles/g scierot sclero.f Control 153 12* 3.8 .3 2.5 .2* mg/g Dilantin 274 15 3.5 .3 6.1 .8 Control 40 4* 3.8 4*.09 Cmpd. I 156 11* 3.6 .2 2.9 .2* .01* Dilantin 210 20 1 .3 .1.16 .01 Cmpd. II 133 10* 4.7 4*3.2 .2* Cmpd. I 70 8* 2.1 .2* .05 .01* Cmpd. III 112 9* 3.0 .2 2.8 .2* Cmpd. II 122 12* 1.1 .1 Cmpd. IV 206 10* 3.9 .2 3.7 .2* .04 .01* Cmpd. III 10* 0.98 .1*.03 .01* Cmpd. V 135 10* 3.6 .2 2.2 .2* 114 Cmpd. IV 88 9* 2.3 .2* .01* Normal 207 15*3.0 .2 2.9 3* .04 Cmpd. V 35 4* 3.9 4*.11 .01* * Significantlydifferent from Dilantin (p < Normal 18 2* 11.5 1*.14 .01 .05). * Significantlydifferent from Dilantin (p < t Hypro =hydroxyproline;Hex =hexosamine; .05). N =nitrogen. = Italicizedmeans are significantly different from f Hypro hydroxyprolinein imoles/mg control (p < .05). scieroprotein; Hex =hexosaminein ,moles/mg scleroprotein. Italicized means are significantly different derivatives equivalent to the concentration offrom control (p < .05). Dilantin usually employed did not produce the same kind of changes in these components of the connective tissue, either quantitatively or qual-collagen content of the dermis need not neces- itatively as did Dilantin. Some of the compounds,sarily be associated with increases in scleropro- particularly IV, did result in significant increasestein concentration. above the control in the concentration of in- Since ten daily intraperitoneal injections of one soluble collagen, however. It has been notedml of isotonic saline into 250g rats have been previously (11) that changes in the dermal con-shown to result in a decrease in the concentration centration of collagen and scieroprotein can pro-of total and insoluble collagen (4), it is interesting ceed independently with Dilantin administration.that animals treated in this manner with com- Therefore, it is possible that increases in thepounds I, IV and V did not demonstrate losses CHEMICAL RESPONSE TO DILANTIN ANALOGUES 93 of total collagen from the skin. However, onlyprecise structure of Dilantin rather than upon compound IV was not associated with a loss ofhydantoin-like structures in general. insoluble collagen from the tissue. Despite this "collagenizing" effect of compound REFERENCES IV, it is obvious that none of the analogues of 1. Zrsirc, D. E., STOWE, L. 14. AND ZEGARELL!, E. V.: Dilantin hyperplastic gingivitis its Dilantin studied could quantitatively equal all cause and treatment, a different appraisal. the changes in connective tissue chemistry pro- Amer. J. Orthodont., 27: 350, 1941. 2. Houcx, J. C., JAcoB, 14. A. AND MAENGWvN- duced by Dilantin itself. Therefore these ana- DAvIEs, C. D.: The effect of sodium Dilantin logues either did not possess the same biological administration upon the chemistry of the properties as Dilantin, or their activities in these skin. J. Clin. Invest., 39: 1758, 1960. 3. SHAFER, W. C.: Effect of Dilantin sodium directions were sufficiently small so that when analogues on cell proliferation in tissue employed at doses equivalent to that of Dilantin culture. Proc. Soc. Exp. Biol. Med., 106: 205, they were not demonstrable. 1961. 4. SETH!, P. AND bucK, J. C.: Dermal collagen response to injury. J. Invest. Derm., 37: 85, 5UMMAEY 1961. 5. Houcx, J. C. AND JAcoB, R. A.: Chemical Five derivatives of Dilantin, including two dissection of rat skin. Proc. Soc. Exp. Biol. Med., 105: 324, 1960. compounds which, like Dilantin, have been shown 6. JAcoB, II. A. AND bucK, J. C.: A chemical to accelerate the proliferation of fibroblasts in description of inflammation. Surg. Gynec. Obstet., 109: 85, 1959. tissue culture (3), were found not to be able to 7. BLix, C.: The determination of hexosamines produce all the alterations in dermal chemistry according to Elson and Morgan. Acta. Chem. seen following Dilantin administration (2). The Scand., 2: 467, 1948. 8. MARTIN, C. J. AND AXELEOD, A. E.: A modified administration of one of these derivatives, method for the determination of hydroxy- 1-allyl-5 phenyl hydantoin to whole animals re- proline. Proc. Soc. Exp. Biol. Med., 83: 461, 1953. sulted in a significant increase in the dermal 9. Bo&s, N. F.: Distribution of hexosamine in concentration of insoluble collagen, but this electrophoretically separated extracts of rat effect was considerably less than that produced connective tissue. Arch. Biochem., 57: 367, 1955. by similar amounts of Dilantin and the other10. NEUMAN, 14. E. AND LOGAN, M. A.: The dermal chemical responses to Dilantin adminis- determination of collagen and elastin in tissues. J. Biol. Chem., 186: 549, 1950. tration were not demonstrated by this drug. 11. boucx, J. C.: The resorption of Dilantin These results suggest that 1) increased fibro- produced dermal collagen. J. Clin. Invest., blast proliferation alone does not explain the 41:179, 1962. effects of Dilantin upon the connective tissue and 12. CHUNG,A.C. ANDHoucK, J. C.:The dermal lipidresponse to Dilantin. Proc. Soc. Exp. 2) that these effects depend upon the unique and Biol. Med., 109: 454, 1962.