WALTER M . D A L E 299 EFFECTS OF RADIATIONS ON AQUEOUS SOLUTIONS OF INDOLE BY C. B. ALLSOPP AND MISS J. WILSON Guy’s Hospital Medical School London S.E. 1 Received 24th January 1952 As estimated with Ehrlich’s reagent indole appears to be readily decomposed by small doses of X-radiation (i.e. up to 20 r). The quantity decomposed increases with (i) in- creasing concentration of indole and (ii) decreasing dosage-rate. The initial decomposi- tion is rather greater in solutions prepared and irradiated in an atmosphere of nitrogen than in aerated solutions the subsequent reaction appears to be quantitatively the same. The radiation-chemical and photo-chemical decompositions of indole (in presence of air) follow similar courses. These effects are considered to be consistent with an initial oxidation to indole-5 6-quinone followed by oxidative condensation of this substance with unchanged indole.Preliminary accounts have already been given of the sensitivity of aqueous solutions of indole to gamma rays 1 and to soft X-rays.2 The results described here include the effects of X-rays half-value layer 1.2 mm of aluminium over the concentration range from 3-0 to 130 mg/l. of indole and the dosage-rate range from 2 to 63 rontgens/min in the presence and absence of oxygen together with some observations on the effects of monochromatic radiation of wave-length 2537A on indole solutions exposed to air. SOLUTIONS OF INDOLE 300 EXPERIMENTAL A N D RESULTS The solutions were exposed to X-rays in small stoppered glass tubes containing 1 rnl under conditions already described.2 Indole was estimated absorptiometrically with Ehrlich’s reagent (pdirnethylaminobenzaldehyde).3 The X-ray tube is air-cooled and has a maximum running time of 10 min before becoming overheated this factor limited the range of both dose and dosage-rate which could conveniently be employed.Since the irradiations were made under standardized conditions throughout the doses are expressed in rontgens as measured in air with a Victoreen dosemeter. They are propor- tional to the energy dissipated in the irradiated solutions. Each point on the curves of fig. 1 to 5 represents the mean of at least six separate observations and the statistical error is covered by the size of each point. VARIATION OF THE QUANTITY OF INDOLE DECOMPOSED WITH X-RAY DOSE.-Fig.1 Shows two curves representing the largest and smallest effects which we have observed which relate the quantity of indole changed per rontgen to the total dose in rontgens. These curves are characteristic of all the experiments. For very small doses there appears to FIG. 1.-Variation of quantity of indole changed per rontgen with total dose in rijntgens (a) indole concentration 100 mg/l. ; dose-rate 2.8 r/min ; (6) indole concentration 30 mg/l. ; dose-rate 70 r/min. be a rapid decomposition but the quantity decomposed per rontgen falls off at first rapidly and later for doses above about 10 r more slowly. Over the range of doses studied it never becomes constant as it should if at any stage the quantity decomposed were proportional to the total dose.The initial parts of the curves correspond to the decomposition of several hundred molecules of indole for each ion-pair formed in the solution. This result has been confirmed by direct measurements of the change in intensity of the absorption band of indole at 2700 8 during irradiation. The mean of three spectro- photometric determinations of the decomposition in a solution containing 130 mg/l. caused by 2-8 delivered in 1 min was 6.2 % as compared with 7.2 % estimated absorptio- metrically with Ehrlich’s reagent. The difference is less than the experimental error in the spectrophotometric determination. EFFECT OF DOSAGE RATE.-The effect of dosage rate is shown in fig. 2 in which the quantity of indole decomposed by a dose of 1 r is plotted against dosage rate in r per min for a solution containing 30 mg/l.of indole. The decomposition is greatest at the lowest dosage rates. It decreases by about 80 % between rates of 2 and 40 r/min but then appears to be approaching a constant value. EFFECT OF CONCENTRATION.-NO simple quantitative relationship has been deduced between the decomposition observed at the smallest dose measured and the concentration of indole although the graph relating them is a smooth curve. In fig. 3 it is plotted for convenience on logarithmic scales. The maximum decomposition of indole per rontgen = 1 r. C . B . ALLSOPP AND MISS J. WILSON increases very rapidly with the concentration in the curve shown by a factor of 100 over the concentration range studied.EFFECT OF OXYGEN.-In the experiments described so far no attempt was made to exclude oxygen from the solutions. Experiments have however been made in solutions in which the pressure of oxygen was reduced. For this purpose the manipulations were FIG. 2. - Variation with dosage-rate of the quantity of indole changed by one rontgen indole concentra- tion = 30 mg/l. ; total dose FIG. 3.-Variation with concentration of the quantity of indole changed per rontgen dosage-rate = 2-8 r/niin ; total dose = 2.8 r. I .?.5 x 10-5 carried out by remote control under a sealed bell-jar in an atmosphere of nitrogen. The indole was previously weighed into a graduated flask and placed under the bell-jar so that air could be swept out by a current of nitrogen before the oxygen-free water was brought to it.Nitrogen was bubbled through the solution in the atmosphere of nitrogen until all the indole was dissolved which required about four days. 1 rnl portions were trans- ferred to irradiation tubes which were stoppered all without any exposure to air. The nitrogen-filled solutions were irradiated simultaneously with ordinary solutions and the irradiated specimens were analyzed side by side. K* 301 302 SOLUTIONS OF INDOLE Typical results are shown in fig. 4. For very small doses the quantity of indole de- composed was always a few per cent higher at the reduced oxygen pressure at higher doses there was little significant difference. The nature of this difference did not seem to FIG.4.-Effect of excluding oxygen on X-ray decomposition of indole indole concentration = 30 mg/l. Full line = oxygen present ; broken line = irradiation in nitrogen ; dosage-rate (a) 70 r/min ; (6) 25 r/min. fild dose punto absorbed ,200 I iQ0 xi0 IS FIG. 5.-Photochemical decomposition of indole indole concentration = 30 mg/l. quanta absorbed per second under each cm2 of exposed surface (a) 1.0 x 1014; (b) 4.0 x 1014. justify any more elaborate measures to exclude oxygen. It is most probably due to in- creased stability of the unirradiated indole solution in the absence of oxygen indole being known to undergo slow auto-oxidation. No significant difference was observed between the effects produced when the indole solutions were irradiated simultaneously in Pyrex and fused silica tubes respectively.303 PHOTOCHEMISTRY OF INDOLE.-The solutions were exposed to various intensities of ultra-violet radiation X = 2537A in shallow layers in open Petri dishes placed under a neon-sensitized mercury lamp 4 under conditions described elsewhere.5 Incident light intensities in quanta/cm2 sec were measured by the uranyl oxalate actinometer method of Leighton and Forbes.6 The results from two experiments are shown in fig. 5 where for comparison with fig. 1 the decomposition is plotted as milligrams of indole decomposed per 1020 quanta absorbed against the total number of quanta absorbed this latter number being deduced from the incident intensity the molecular extinction coefficient E = 1000 at X = 2537 A,7 and the geometry of the irradiated solution.The general form of the curves is similar to those of fig 1 and they have the same feature that the greater decom- position is caused at the lower intensity. No smooth curve corresponding to fig. 2 has however been obtained in the photochemical experiments. (IV) isatin (I) indole C. R. ALLSOPP AND MISS J . WILSON (11) indoxyl (V) indole 5 6-quinone DISCUSSION In interpreting these results the following considerations are relevant (i) Ehrlich’s reagent is not specific for indole but is a general reagent for pyrrole groups; the almost steady concentration which is recorded at the higher X-ray doses thus indicates that the final product of the chemical reactions contains sub- stantially unchanged pyrrole.(ii) At the concentrations which have been used the action of the X-rays must be indirect and the chemical reactions are most probably initiated through the production of hydroxyl and hydrogen radicals from the water ; in the presence of oxygen other oxidizing radicals may also be present. (iii) In irradiated solutions the effect of oxidizing radicals usually pre- dominates 8 and the reactions now observed may be expected to be oxidative in character. (iv) The very large ionic yields in the early stages of the reactions suggest that some form of chain mechanism is involved. Indole can be oxidized either on the pyrrole ring e.g. at the 2- or 3-positions or on the phenyl ring at the 5- or 6-positions. An investigation of the first alter- native was made possible by a gift of materials from I.C.I.Ltd. Indole (I) and indoxyl (11) gave red colours with Ehrlich’s reagent. Dioxindole (111) prepared from isatin by reduction with alkaline sodium hydrosulphite,9 and isatin (IV) did not. The ability to produce the characteristic red colour with Ehrlichs reagent disappears with the double bond between the 2- and 3-carbon atoms. Indoxyl is extremely unstable and its formation could not possibly account for the apparent stability of indole to larger radiation doses. The oxidation deriva- tives of indole in the 2- and 3-positions readily oxidize to indigo; but no blue colour has been observed in any irradiated solution. It seems improbable therefore that this type of oxidation could account for our observations.(111) dioxindole Indole derivatives in which oxidation has taken place at the 5- or 6-positions as in the quinone (V) occur during the enzymatic oxidation of tyrosine,lo and there is spectroscopic evidence that they are also present among the products of photochemical oxidation of tyrosine.7 Harley-Mason 11 has described reactions between quinones and indoles to form compounds such as 4 3’-indolyl-1 2- benzoquinone (VI) the condensation occurring at the 3-position. He has put (VI) 4 3’4ndolyl-1 2-benzoquinone 304 forward strong evidence that a melanin may be produced by polymerization of indole-5 6-quinone (V) by repeated oxidative condensation involving the 3- position of one molecule and the 4- or 7-positions of another as in (VII).Tt is significant that such a polymerization would not destroy the pyrrole rings and so reaction with Ehrlich's reagent would still be possible although we have not as yet been able to test this. As the size of the molecule increases the nature of the Ehrlich colour would be slightly modified such changes were described in our previous paper.' To explain our present results therefore we suggest that oxidation of indole to 5 6-dihydroxy-indole or to indole-5 6-quinone might perhaps be the first stage in these radiation-induced reactions and that this is followed by polymer- ization. The largest effect is produced at lowest intensities of X-rays and at highest concentrations. A low intensity of X-rays would produce a low initial concentra- tion of hydroxyl radicals and the oxidized molecules would be formed relatively far apart from one another.The most likely polymerization since indole seems to disappear most at higher concentrations would then be an oxidative condensa- tion of the quinone with unchanged indole which could be propagated as in (VII) although condensation of quinone with quinone is not excluded. The suggestion is a tentative one and much further work will be necessary before this mechanism can be confirmed but as a working hypothesis it does not appear to be incon- sistent with the observations so far recorded. From the biological viewpoint the explanation is attractive in that it suggests that the pigmentation resulting from exposure to ionizing radiations may originate in the chemical degeneration of protein leading ultimately to melanin formation.Our study of the effects of X-rays on other indole compounds may throw light on this. Our results also suggest that it might be of value to re-examine with low intensities and low doses of ionizing radiation some of the substances of biological interest which appear to be " radiation resistant " when subjected to large intense doses. These experiments form part of a programme of research financed by the British Empire Cancer Campaign to which body the authors express their thanks. They are also indebted to I.C.I. Ltd. for a gift of materials and to Dr. M. A. T. Rogers and to Dr. F. H. Brain for advice on the preparation of indoxyl and dioxindole. 4 Melville Trans. Faraday Soc.1936 32 1525. SOLUTIONS OF INDOLE (VII) suggested polyrncrization of indole-5 6-quinone (Harley -Mason) 1 Allsopp Medical R4.r. Corrricil Reports (Metlical U.vcs of Rarlirrni) 1937 no. 2.32 12 ; 1938 no. 236 1 I. 2 Allsopp and Wilson f. Cfiiin. Phys. 1951 48 195. 3 Allsopp Biocfiem. f. 1941 35 965. 5 Allsopp and Szigeti Cancer Res. 1946 6 14. 305 C . B . ALLSOPP AND MISS J . WILSON 6 Leighton and Forbes J . Ameu. Chern. SOC. 1930 52 3139. 7 Allsopp unpublished work. 8 see for instance Haissinsky and Lefort Cmnpt. rend. 1950 230 1156. 9 Maschalk Rer. 1912 45 582. 10 see Raper J . Chem. SOC. 1938 1952. 11 see Bu’Lock and Harley-Mason J. C h n . Suc. 1951 703. WALTER M . D A L E 299 EFFECTS OF RADIATIONS ON AQUEOUS SOLUTIONS OF INDOLE BY C.B. ALLSOPP AND MISS J. WILSON Guy’s Hospital Medical School London S.E. 1 Received 24th January 1952 As estimated with Ehrlich’s reagent indole appears to be readily decomposed by small doses of X-radiation (i.e. up to 20 r). The quantity decomposed increases with (i) in-creasing concentration of indole and (ii) decreasing dosage-rate. The initial decomposi-tion is rather greater in solutions prepared and irradiated in an atmosphere of nitrogen than in aerated solutions the subsequent reaction appears to be quantitatively the same. The radiation-chemical and photo-chemical decompositions of indole (in presence of air) follow similar courses. These effects are considered to be consistent with an initial oxidation to indole-5 6-quinone followed by oxidative condensation of this substance with unchanged indole.Preliminary accounts have already been given of the sensitivity of aqueous solutions of indole to gamma rays 1 and to soft X-rays.2 The results described here include the effects of X-rays half-value layer 1.2 mm of aluminium over the concentration range from 3-0 to 130 mg/l. of indole and the dosage-rate range from 2 to 63 rontgens/min in the presence and absence of oxygen together with some observations on the effects of monochromatic radiation of wave-length 2537A on indole solutions exposed to air 300 SOLUTIONS OF INDOLE EXPERIMENTAL A N D RESULTS The solutions were exposed to X-rays in small stoppered glass tubes containing 1 rnl under conditions already described.2 Indole was estimated absorptiometrically with Ehrlich’s reagent (pdirnethylaminobenzaldehyde).3 The X-ray tube is air-cooled and has a maximum running time of 10 min before becoming overheated this factor limited the range of both dose and dosage-rate which could conveniently be employed.Since the irradiations were made under standardized conditions throughout the doses are expressed in rontgens as measured in air with a Victoreen dosemeter. They are propor-tional to the energy dissipated in the irradiated solutions. Each point on the curves of fig. 1 to 5 represents the mean of at least six separate observations and the statistical error is covered by the size of each point. two curves representing the largest and smallest effects which we have observed which relate the quantity of indole changed per rontgen to the total dose in rontgens.These curves are characteristic of all the experiments. For very small doses there appears to VARIATION OF THE QUANTITY OF INDOLE DECOMPOSED WITH X-RAY DOSE.-Fig. 1 Shows FIG. 1.-Variation of quantity of indole changed per rontgen with total dose in rijntgens : (a) indole concentration 100 mg/l. ; dose-rate 2.8 r/min ; (6) indole concentration 30 mg/l. ; dose-rate 70 r/min. be a rapid decomposition but the quantity decomposed per rontgen falls off at first rapidly and later for doses above about 10 r more slowly. Over the range of doses studied it never becomes constant as it should if at any stage the quantity decomposed were proportional to the total dose. The initial parts of the curves correspond to the decomposition of several hundred molecules of indole for each ion-pair formed in the solution.This result has been confirmed by direct measurements of the change in intensity of the absorption band of indole at 2700 8 during irradiation. The mean of three spectro-photometric determinations of the decomposition in a solution containing 130 mg/l. caused by 2-8 delivered in 1 min was 6.2 % as compared with 7.2 % estimated absorptio-metrically with Ehrlich’s reagent. The difference is less than the experimental error in the spectrophotometric determination. EFFECT OF DOSAGE RATE.-The effect of dosage rate is shown in fig. 2 in which the quantity of indole decomposed by a dose of 1 r is plotted against dosage rate in r per min for a solution containing 30 mg/l. of indole. The decomposition is greatest at the lowest dosage rates.It decreases by about 80 % between rates of 2 and 40 r/min but then appears to be approaching a constant value. EFFECT OF CONCENTRATION.-NO simple quantitative relationship has been deduced between the decomposition observed at the smallest dose measured and the concentration of indole although the graph relating them is a smooth curve. In fig. 3 it is plotted for convenience on logarithmic scales. The maximum decomposition of indole per rontge C . B . ALLSOPP AND MISS J. WILSON 301 increases very rapidly with the concentration in the curve shown by a factor of 100 over the concentration range studied. EFFECT OF OXYGEN.-In the experiments described so far no attempt was made to exclude oxygen from the solutions.Experiments have however been made in solutions in which the pressure of oxygen was reduced. For this purpose the manipulations were FIG. 2. - Variation with dosage-rate of the quantity of indole changed by one rontgen indole concentra-tion = 30 mg/l. ; total dose = 1 r. .?.5 x 10-5 I FIG. 3.-Variation with concentration of the quantity of indole changed per rontgen dosage-rate = 2-8 r/niin ; total dose = 2.8 r. carried out by remote control under a sealed bell-jar in an atmosphere of nitrogen. The indole was previously weighed into a graduated flask and placed under the bell-jar so that air could be swept out by a current of nitrogen before the oxygen-free water was brought to it. Nitrogen was bubbled through the solution in the atmosphere of nitrogen until all the indole was dissolved which required about four days.1 rnl portions were trans-ferred to irradiation tubes which were stoppered all without any exposure to air. The nitrogen-filled solutions were irradiated simultaneously with ordinary solutions and the irradiated specimens were analyzed side by side. K 302 SOLUTIONS OF INDOLE Typical results are shown in fig. 4. For very small doses the quantity of indole de-composed was always a few per cent higher at the reduced oxygen pressure at higher doses there was little significant difference. The nature of this difference did not seem to FIG. 4.-Effect of excluding oxygen on X-ray decomposition of indole : indole concentration = 30 mg/l. Full line = oxygen present ; broken line = irradiation in nitrogen ; dosage-rate (a) 70 r/min ; (6) 25 r/min.fild dose punto absorbed I iQ0 ,200 xi0 IS FIG. 5.-Photochemical decomposition of indole : indole concentration = 30 mg/l. quanta absorbed per second under each cm2 of exposed surface : (a) 1.0 x 1014; (b) 4.0 x 1014. justify any more elaborate measures to exclude oxygen. It is most probably due to in-creased stability of the unirradiated indole solution in the absence of oxygen indole being known to undergo slow auto-oxidation. No significant difference was observed between the effects produced when the indole solutions were irradiated simultaneously in Pyrex and fused silica tubes respectively C. R. ALLSOPP AND MISS J . WILSON 303 PHOTOCHEMISTRY OF INDOLE.-The solutions were exposed to various intensities of ultra-violet radiation X = 2537A in shallow layers in open Petri dishes placed under a neon-sensitized mercury lamp 4 under conditions described elsewhere.5 Incident light intensities in quanta/cm2 sec were measured by the uranyl oxalate actinometer method of Leighton and Forbes.6 The results from two experiments are shown in fig.5 where for comparison with fig. 1 the decomposition is plotted as milligrams of indole decomposed per 1020 quanta absorbed against the total number of quanta absorbed this latter number being deduced from the incident intensity the molecular extinction coefficient E = 1000, at X = 2537 A,7 and the geometry of the irradiated solution. The general form of the curves is similar to those of fig 1 and they have the same feature that the greater decom-position is caused at the lower intensity.No smooth curve corresponding to fig. 2 has, however been obtained in the photochemical experiments. DISCUSSION In interpreting these results the following considerations are relevant : (i) Ehrlich’s reagent is not specific for indole but is a general reagent for pyrrole groups; the almost steady concentration which is recorded at the higher X-ray doses thus indicates that the final product of the chemical reactions contains sub-stantially unchanged pyrrole. (ii) At the concentrations which have been used, the action of the X-rays must be indirect and the chemical reactions are most probably initiated through the production of hydroxyl and hydrogen radicals from the water ; in the presence of oxygen other oxidizing radicals may also be present.(iii) In irradiated solutions the effect of oxidizing radicals usually pre-dominates 8 and the reactions now observed may be expected to be oxidative in character. (iv) The very large ionic yields in the early stages of the reactions suggest that some form of chain mechanism is involved. Indole can be oxidized either on the pyrrole ring e.g. at the 2- or 3-positions, or on the phenyl ring at the 5- or 6-positions. An investigation of the first alter-native was made possible by a gift of materials from I.C.I. Ltd. Indole (I) and indoxyl (11) gave red colours with Ehrlich’s reagent. Dioxindole (111) prepared from isatin by reduction with alkaline sodium hydrosulphite,9 and isatin (IV) did not. The ability to produce the characteristic red colour with Ehrlichs reagent disappears with the double bond between the 2- and 3-carbon atoms.Indoxyl is extremely unstable and its formation could not possibly account for the apparent stability of indole to larger radiation doses. The oxidation deriva-tives of indole in the 2- and 3-positions readily oxidize to indigo; but no blue colour has been observed in any irradiated solution. It seems improbable therefore that this type of oxidation could account for our observations. (I) indole (11) indoxyl (111) dioxindole (IV) isatin Indole derivatives in which oxidation has taken place at the 5- or 6-positions, as in the quinone (V) occur during the enzymatic oxidation of tyrosine,lo and there is spectroscopic evidence that they are also present among the products of photochemical oxidation of tyrosine.7 Harley-Mason 11 has described reactions between quinones and indoles to form compounds such as 4 3’-indolyl-1 2-benzoquinone (VI) the condensation occurring at the 3-position.He has put (V) indole 5 6-quinone (VI) 4 3’4ndolyl-1 2-benzoquinon 304 SOLUTIONS OF INDOLE forward strong evidence that a melanin may be produced by polymerization of indole-5 6-quinone (V) by repeated oxidative condensation involving the 3-position of one molecule and the 4- or 7-positions of another as in (VII). Tt is significant that such a polymerization would not destroy the pyrrole rings and so reaction with Ehrlich's reagent would still be possible although we have not as yet of the in our To been able to test this.As the size of the molecule increases the nature Ehrlich colour would be slightly modified such changes were described previous paper.' (VII) suggested polyrncrization of indole-5 6-quinone (Harley -Mason) explain our present results therefore we suggest that oxidation of indole to 5 6-dihydroxy-indole or to indole-5 6-quinone might perhaps be the first stage in these radiation-induced reactions and that this is followed by polymer-ization. The largest effect is produced at lowest intensities of X-rays and at highest concentrations. A low intensity of X-rays would produce a low initial concentra-tion of hydroxyl radicals and the oxidized molecules would be formed relatively far apart from one another. The most likely polymerization since indole seems to disappear most at higher concentrations would then be an oxidative condensa-tion of the quinone with unchanged indole which could be propagated as in (VII), although condensation of quinone with quinone is not excluded.The suggestion is a tentative one and much further work will be necessary before this mechanism can be confirmed but as a working hypothesis it does not appear to be incon-sistent with the observations so far recorded. From the biological viewpoint the explanation is attractive in that it suggests that the pigmentation resulting from exposure to ionizing radiations may originate in the chemical degeneration of protein leading ultimately to melanin formation. Our study of the effects of X-rays on other indole compounds may throw light on this. Our results also suggest that it might be of value to re-examine with low intensities and low doses of ionizing radiation some of the substances of biological interest which appear to be " radiation resistant " when subjected to large intense doses. These experiments form part of a programme of research financed by the British Empire Cancer Campaign to which body the authors express their thanks. They are also indebted to I.C.I. Ltd. for a gift of materials and to Dr. M. A. T. Rogers and to Dr. F. H. Brain for advice on the preparation of indoxyl and dioxindole. 1 Allsopp Medical R4.r. Corrricil Reports (Metlical U.vcs of Rarlirrni) 1937 no. 2.32 12 ; 2 Allsopp and Wilson f. Cfiiin. Phys. 1951 48 195. 3 Allsopp Biocfiem. f. 1941 35 965. 4 Melville Trans. Faraday Soc. 1936 32 1525. 5 Allsopp and Szigeti Cancer Res. 1946 6 14. 1938 no. 236 1 I C . B . ALLSOPP AND MISS J . WILSON 6 Leighton and Forbes J . Ameu. Chern. SOC. 1930 52 3139. 7 Allsopp unpublished work. 8 see for instance Haissinsky and Lefort Cmnpt. rend. 1950 230 1156. 9 Maschalk Rer. 1912 45 582. 10 see Raper J . Chem. SOC. 1938 1952. 11 see Bu’Lock and Harley-Mason J. C h n . Suc. 1951 703. 30