Toxicology, 63 (1990) 85--95 Elsevier Scientific Publishers Ireland Ltd.

Photochemical reactions of quindoxin, olaquindox, carbadox and cyadox with protein, indicating photoallergic properties H.

de Vries a, J.

Bojarski b, A . A . Donker a, A. Bakri a and Beyersbergen van Henegouwen a

G.M.J.

~Center f o r Bio-Pharmaceutical Sciences, State University o f Leiden. P.O. Box 9502, 2300 RA Leiden (The Netherlands) and ~Nicolaus Copernicus Academy o f Medicine in Cracow. Dzierz, ynskiego 14 B str., 30--048 Krakow (Poland) (Received December 4th, 1989; accepted March 23rd, 1990)

Summary Quindoxin (quinoxaline-l,4-dioxide), a former 'growth promoter' used in animal husbandry, has been taken from the market because of its photoallergic properties. Nowadays its derivatives olaquindox, carbadox and cyadox are frequently applied for the same purpose. Recent reports show that olaquindox too, can induce photoallergic skin reactions in stockmen. From the present investigation it appeared that all compounds mentioned, form a reactive oxaziridine upon exposure to light, just like many other imino-N-oxides. Photoreactivity with protein, which is considered as an important condition for a compound to be a potential photoallergen, was also studied. Quindoxin and olaquindox proved to meet thi~ condition, as was expected. But carbadox and cyadox also react and were shown to be even more reactive towards human serum albumin.

Key words: Quindoxin; Carbadox; Olaquindox; Cyadox; Growth promoters; Photoallergy; Oxaziridine; Human serum albumin; Isoelectric focusing

Introduction Derivatives of quindoxin (QUIN; quinoxaline-l,4-dioxide), such as olaquindox (OLAQ; e.g. BAY-O-NOX R, Bayer), Carbadox (CARB; e.g. MECADOX R, Pfizer) and Cyadox (CYAD; Chemapol Benelux) (Fig. 1) are feed additives, frequently used in stock-raising to improve growth of amongst others, piglets [1-31. QUIN itself has been used for the same purpose but was taken from the market in 1973 because it caused persistent photocontact dermatitis in several stockmen [4]. All the patients suffering from the dermatitis had been handling animal feeding stuffs. Direct contact of the growth promoter with skin (hands mostly) is supposed to be the 'administration route'. The acute symptoms are Address all correspondence and reprint requests to: H. de Vries. 0300-483X/90/$03.50 c~j 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

85

QUINDOXIN :

RI=R2:H

0 {~i/~.

I

H R1

CARBADOX :

RI: - C H = N - N-C--OCH3

R2

CYA D0X :

R2= H H 0 It, R1 = - CH=N - N --(~/- CH2CN R2= H

0 OLAQUINDOX :

OH // I

R I = - C - N--CH2-CH20H R2= - CH3

Fig. l.Molecular structure of QUINdoxin and its derivatives OLAQuindox, CARBadox and CYADox.

mostly a dermatitis of the exposed skin and an abnormally high sensitivity to UV-B (290--320 nm) and in some cases to UV-A (320--400 rim). Light sensitivity lasted in many cases for more than 5 years [4]. More recently the quindoxin derived OLAQ has also been reported to cause allergy [5] and photoallergy in man [6]. Symptoms of OLAQ-induced photoallergy [6] resemble those of QUIN [7]. Generally, a photochemical reaction of a xenobiotic resulting in damage of, or irreversible binding to an endogenous protein, is considered as an initial step which may lead to a photoallergic response [8--10]. The feed additives mentioned belong to the group of imino-N-oxides of which some representatives are known to be able to form oxaziridines upon exposure to sunlight (Fig. 2); the latter appeared to be very reactive with proteins Ill]. It was investigated whether the growth promoters also form an oxaziridine upon UV irradiation. In connection with the possible formation o f oxaziridines, the photoreactivity of QUIN, OLAQ, CARB and CYAD with protein was studied. Results with photoallergens QUIN and OLAQ were compared with those for CARB and CYAD of which adverse photobiological effects have not (yet) been reported in the literature. Materials and methods

Demineralised water was distilled in an all-glass apparatus before use. Organic solvents were 'chemically pure' and used after distillation. Phosphate buffered saline (PBS) was made by dissolving 4 g NaCI, 7.5 g Na2HPO4.2H20 and 3 g KH2PO 4 (all from Merck, Darmstadt, F.R.G.) in I 1 water. Olaquindox (OLAQ),

~C=N/ h~'~C-Ni T P > ~ = ~ 0

0

Fig. 2. Photoisomerisation of an imino-N-oxide into an oxaziridine, followed by reduction of the latter by TP.

86

carbadox (CARB), and cyadox (CYAD) were a gift from Dr. A.J. Baars (Central Veterinary Institute, Lelystad, The Netherlands) and used after recrystallisation from acetone. Quindoxin (QUIN) was synthesised according to Klein and Berkowitz [12]. Desoxyolaquindox-N4-monoxide, desoxyolaquindox-Nl°monoxide and desoxyolaquindox were a gift from Bayer AG (Leverkusen, F.R.G.) and used as such. Human serum albumin (HSA) came from Sigma (St. Louis, USA). Triphenyl phosphine (TP) was from Merck and used as such.

Reaction with triphenyl phosphine To investigate whether oxaziridines are formed during the photodegradation, samples (1 ml) with growth promoter in the presence or absence of triphenyl phosphine (TP) were exposed to UV-A as described below. The concentrations of growth promoter and of TP, both in methanol, were 0.1 mg/ml (approx. 4 x 10 -4 M for QUIN approx. 6 x 10 -4 M) and 0.5 mg/ml (approx. 2 x 10 -3 M), respectively. HPLC analysis was performed as described below, but as a detector a 1040 A diode array (Hewlett Packard, Amsterdam, The Netherlands) was used, recording spectra between 240 and 450 nm. The HPLC system mentioned was also used for the analysis of photoproducts from CARB, except that in this case a Finnigan MAT TSQ-70 mass spectrometer, equipped with a Finnigan MAT thermospray, replaced the UV-Vis monitor. In these analyses of the HPLC peaks of interest, 50 mM ammonium acetate was added to the eluant to facilitate ionisation. Reaction with HSA The solutions to be irradiated consisted of 0.1 mg/ml (approx. 4 x 10 -4 M; for QUIN approx. 6 x 10 -4 M) of one of the growth promoters mentioned and 2 mg/ml (approx. 3 × 10 -5 M) HSA in PBS with 2.5070 dimethylsulfoxide. Before and after irradiation, solutions were kept in the dark or treated in a dimly lighted environment. For irradiations, Philips TL 80 W/10R lamps were used (spectral region 345-410 nm, ~'m~ 370 rim; a gift from C.C.E. Meulemans M.Sc., Philips Central Lighting Division, Eindhoven, The Netherlands). Samples (1 ml) were exposed to UV-A in glass vials of 11.5 mm diameter (ATS, Waddinxveen, The Netherlands) with a cutoff at 290 nm. The intensity of the light in the proximity of the vials was 150 W /m 2, measured with a UVX radiometer (Ultraviolet Products Ltd. San Gabriel, CA), equipped with a UVX-36 sensor. Solutions were dialysed for 48 h in a "Dianorm R'' device, (Diachema AG, Langnau, Zurich; mol. wt. cut-off of 5000 ) against a bulk volume of water which was regularly exchanged. Concentration of the compounds under investigation was determined by HPLC analysis using an LKB 2150 pump (LKB, Bromma, Sweden) equipped with a Promiss autosampler (Spark, Emmen, The Netherlands), an RP 18 Lichrosorb 7/a 100 × 4 mm column (Chrompack, Middelburg, The Netherlands) and an LKB 2151 variable wavelength UV-Vis monitor set at 370 nm. Chromatograms were recorded and processed by a Shimadzu C-R3A integrator (Shimadzu, Kyoto, Japan). The eluant was methanol/water; for QUIN and OLAQ the methanol 87

content was 12070 (v/v) and for CARB and CYAD 32070 (v/v); flow was 0.5 ml/ min in all cases. Isoelectric focusing was performed with a 2117 multiphor II (LKB) in combination with an LKB 2197 power supply. After irradiation, samples were diluted 4 × with PBS and 10-tal samples were applied to an Ampholine PAG plate; pH range was 4.0--6.5 (LKB). The focussing took 2.5 h with power supply settings of E = 2000 V, I = 25 mA and P = 25 W. Temperature was 3°C. After focussing, the samples were fixed and stained according to the LKB data sheet provided with the PAG plates. The coomassie blue coloured samples were quantified by means of an Ultroscan X L laser densitometer (LKB). Gel filtration was performed using the same solvent delivery system as used for quantitative H P L C analysis. Irradiated samples (200 tal) were injected onto a 300 x 8 mm GlasPac TSK G3000 SW column (LKB) and eluted with PBS (0.75 ml/min ). As a detector, a 1040 A diode array (Hewlett Packard; Amsterdam, The Netherlands) was used. Results

Irradiation o f growth promoters whether or not in the presence o f TP We found that UV-A irradiation (20 min) of QUIN in the presence of TP, eventually results in the formation of desoxyQUIN (dQUIN = quinoxaline). The formation of dQUIN was confirmed by H P L C analysis and comparison of the retention time and UV spectrum with those of an authentic sample of this compound. In the same way, comparison o f H P L C and UV spectral data of photoproducts with those of authentic samples, the photoreaction of OLAQ in the presence of T P was analysed. It appeared that the first photoproducts are the desoxyolaquindox-Nl-monoxide ( d O L A Q n l ) and the desoxyolaquindox-N4-monoxide (dOLAQn4, retention time 4.8 and 6.1 min, respectively). During this photoreaction the concentration of these two products reaches a maximum (at about 3 and about 5 min, respectively). After this, the concentration of the monoxides gradually decreases in favour of that o f desoxyOLAQ (dOLAQ; retention time 15.3 min). dOLAQ, o f which the concentration increases from the beginning of the irradiation, is the only end-product detectable; it reaches its maximum when both monoxides are not detectable anymore, after about 28 min of irradiation. A typical H P L C chromatogram (Fig. 3) of CARB and its photoproducts, formed in the presence o f TP, reveals three major product " p e a k s " of which two appeared to have a mass of M-16 (retention time 3.7 and 5.8 min ) and one of M32 (retention time 17.9 rain ). The first two were assumed to be the NI- and N4monoxide of desoxyCARB (dCARB) and the third, dCARB. This was further supported by the correspondence of the retention times with those of the photoproducts of OLAQ mentioned previously. The kinetics of the CARB photodegradation nicely correspond to that of OLAQ: an initial increase of the two Nmonoxides (M-16) is followed by a decrease in favour of the end product, dCARB (M-32, see Fig. 3). The mass spectral analyses of the products also revealed an important amount o f triphenyl phosphine oxide.

88

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120 CYAD.

80

100

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20 2(?

0

I

2

3 4 m0n of 0rr

5

6

7

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Fig. 3. Upper panel: Typical H P L C c h r o m a t o g r a m , including retention times, of CARB and CYAD and their photoproducts. Lower panel: H P L C peak heights as a function of irradiation time of CARB and C Y A D and their photoproducts. (Peak heights for C A R B and C Y A D have been recorded at an attenuation of 4 × , in relation to those of the products, to fit them in the figure.) O, CARB (left) and C Y A D (right); * and [I, their desoxy IN- or 4N- monoxides; &, their desoxy c o m p o u n d .

During irradiation o f C Y A D in the presence o f TP, three compounds are formed with retention times o f 3.4, 5.1 and 8.8 min. The latter compound, which is formed almost from the beginning o f the irradiation, has a UV spectrum with maxima at 240, 335 and 396 nm and minima at 270 and 365 nm. The kinetic profile resembles that o f the photodegradation o f C A R B (see Fig. 3).

89

550 pI. 5.25 ~,~

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10

2"0

~

5.00 4.75 40

6(3

100~ % 8060t,0200

'

260

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seconds

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Fig. 4. (a) Representative example of an electropherogram of human serum albumin (2 mg/ml) after UV-A exposure in the presence of a quinoxaline-l,4-diN-oxide (i.c. 0.1 mg/ml carbadox; 150 W/m:, ~..= = 370 rim). The ordinate represents pl values as determined with isoelectric focusing. On the abscissa, irradiation times (min) of the samples are displayed. (b) Decrease of the major band of human serum albumin (2 mg/ml)-isoelectric focusing: pl = 4.9-as a result o f exposure to UV-A light (150 W / m 2, ;'m,, = 370 nm) in the presence of either quindoxin ( + ), olaquindox (r'l), carbadox (Zi) or cyadox ( O ) (each 0.1 mg/ml). The intensity of the 4.9 band, as °/0 of that of HSA in the starting solution, is plotted against the irradiation time.

Identical irradiations were performed with the growth promoters in the absence of TP. Amongst many unknown products, the irradiated samples also contained monoxy and desoxy compounds, as found with TP. But the maximum concentration of these compounds found never exceeded 40°7o of that reached in the presence of TP.

Irradiation of growth promoters with HSA After 400 s (QUIN), 200 s (OLAQ), 40 s (GARB) and 30 s (CYAD) of UV-A irradiation in the presence of growth promoters, the main HSA band (pl = 4.9) as found with isoelectric focusing, was almost completely transformed into several other bands with larger pI (Fig. 4a,b). The electropherogram of HSA did not change after irradiating the protein alone ( + DMSO) or incubating it in the dark with any one of the growth promoters (data not represented). HPLC analysis

90

100 %

80 6040200

0

'

2'0

'

£0

60

seconds

Fig. 5. Photodegradation of quindoxin ( + ) , olaquindox (r-I), carbadox (/x) and cyadox ( ~ ) , in solution with human serum albumin (2 mg/ml), upon exposure to UV-A light (150 W/m 2, ~,~ = 370 rim). Concentrations as a percentage of that of the growth promoter in the starting solution, are plotted against irradiation time.

revealed that during the irradiation 100°70 QUIN, 100°70 OLAQ, 38°70 CARB and 20070 CYAD was decomposed (Fig. 5). Amongst photoproducts of QUIN and OLAQ the corresponding N-monoxides were found, which was evidenced by comparing HPLC and UV-Vis spectral data with those of authentic samples. Irradiating growth promoters in the presence of HSA resulted in changes of the UV spectrum of HSA. With respect to the original albumin spectrum the absorption at 277 nm (HSA max) and at 310 nm (largest change) increased, respectively, with 36070 and 785070 (QUIN; irr. time 400 s), 23070 and 525070 (OLAQ, irr. time 200 s), 4070 and 245070 (CARB; irr. time 40 s) and 27070 and 998070 (CYAD; irr. time 30 s). Protein/growth promoter solutions were kept in the dark, during the irradiation of the others. When these samples were submitted to gel filtration or dialysis afterwards, they had the same UV spectrum as the original HSA. The same holds for an irradiated solution which contained HSA but no growth promoter. Discussion

Both UV-B and UV-A are able to decompose the growth promoters. Photodecomposition with UV-B does not essentially differ from that with UV-A. For instance, when about 50% of growth promoter was decomposed, either with UV-B or UV-A, the same composition of the reaction mixture was obtained (de Vries, Beyersbergen van Henegouwen and de Graaff, unpublished data). Yet, for the following reasons the use of UV-A was preferred. In previous experiments with rats [13], UV-A was used because this kind of radiation as such, is better supported by the skin of these animals than UV-B. Further, without growth promoter being present, UV-B alone already degrades proteins. The aim of the present research was to investigate the effects of the combination of growth promoter/UV-radiation. The use of UV-B would complicate the interpretation of the

91

results obtained. (UV-A alone, does not have any influence on proteins under the experimental conditions applied.) Upon exposure to light, many imino-N-oxides form an oxaziridine (Fig. 2). Because of their three-membered ring structure, these compounds are usually quite unstable and very reactive towards bio(macro)molecules, e.g. serum albumin [I l]. The question arises whether QUIN, OLAQ, CARB and CYAD, all being imino-N-oxides, also primarily form an oxaziridine under the influence of light [14--16] or undergo ring rearrangements only, as is somtimes suggested [17--19]. Kawata et al. [20] described a method to investigate whether an oxaziridine is being formed as an intermediate during a (photo)reaction. When triphenyl phosphine (TP) is present during the formation of an oxaziridine, the oxygen is extracted from the oxaziridine ring, before ring rearrangement or other sequential reactions occur [21,22]. This will result in the formation of a (partly) deoxygenised compound and triphenyl oxyphosphine (TOP). In the case o f the growth promoters, which have two possibilities to form an oxaziridine, the reaction mentioned above will lead to the formation of the monoxides and eventually the desoxy compound. If the growth promoters do not form oxaziridines as an intermediate upon UV-A irradiation but undergo ring rearrangements, oxygen would not be extracted from the molecules by TP and no monoxy or desoxy compounds would be formed to such a high extent. Thus T P was used as a diagnostic to find whether primarily oxaziridines are formed when the growth promoters are irradiated with UV-A. Comparison o f UV-Vis spectral data and retention times of photoreaction products with those of authentic samples and verification of H P L C peak purity was highly facilitated by the computer unit of the photodiode array detector. Irradiation of QUIN in the presence of T P eventually resulted in loss of both oxygen atoms. The photoreaction of OLAQ under the same conditions, was comparable to that of QUIN; both monoxides were found and the endproduct of the irradiation appeared to be dOLAQ. As there were no reference compounds available for CARB, the products formed in the presence of T P were identified by H P L C coupled to a mass spectrometer. CARB was also found to become deoxygenised during irradiation with UV-A in the presence of TP. A confirmation of the fact that T P is taking up oxygen atoms is the finding of T O P in the samples. CYAD is only slightly soluble in almost all common solvents except dimethylsulfoxide. As this solvent is non-compatible with the HPLC-MS technique at our disposal, and as there were no reference compounds available, the nature of the compounds formed upon irradiation in the presence of TP, was identified by comparing the H P L C and UV spectral data with those of the photoproducts from CARB. The reason is that CYAD and CARB only slightly differ with regard to their structure (Fig. 1). As the part of the side chain where the difference is located does not interact with the chromophore, the ring system, the UV spectrum from CYAD is almost identical with that of CARB; both compounds have maxima at 236, 249, 305 and 369 nm and minima at 242, 267 and 337 nm. In addition, the ratios between maximal and minimal extinctions are equal. The

92

latter could easily be observed by comparing the UV spectra of both compounds, which had been normalised by the diode array computer; the spectra of CYAD and CARB almost coincide. Thus it was expected that the N-monoxides and desoxy compounds from CARB and CYAD also have (almost) identical UV spectra. Because of the retention time of the primary photoproducts of CYAD in the presence of TP (3.4 min and 5.1 min), they were supposed to be the N-monoxides of desoxyCYAD (dCYAD; dOLAQ-N-monoxides, 4.8 and 6.1 rain; dCARBN-monoxides, 3.7 and 5.8 min). The kinetics followed were the same as found for OLAQ and CARB: the amount of the two compounds reached a maximum and decreased afterwards in favour of a third product (see Fig. 3). The latter compound, which is formed almost from the beginning of the irradiation, has a retention time of 8.8 rain and a UV spectrum with maxima at 240, 335 and 396 nm and minima at 270 and 365 nm (dCARB: maxima at 240, 334 and 395 nm; minima at 268 and 365 nm). From this detailed correspondence observed between UV spectral data and reaction kinetics, it was concluded that the eventual product of the irradiation of CYAD in the presence of TP is dCYAD. Although the retention time of the precursors of dCYAD (3.4 and 5.1 min) nicely correspond with that of dCARBnl and dCARBn4, this only holds for one of the two products (5.1 min) with regard to the UV data (dCARBnlor4, retention time 5.8 min; max at 280 nm and shoulders at 330, 350 and 368 nm; product from CYAD, 5.1 min, max at 282 nm and shoulders at 330, 350 and 370 nm). As yet we do not have further experimental data to confirm whether the second precursor (3.4 min) of dCYAD is also an N-monoxide. In the presence of TP, the monoxy and desoxy compounds are by far the main products of the irradiation of the four quinoxaline-diN-oxides. In the absence of TP these photoproducts are also formed but their concentration does not exeed 40°70 of that reached in the presence of TP. In the presence of TP, oxaziridines do not get the possibility to undergo secondary reactions like intramolecular rearrangements or dimer formation. The oxygen is taken from the oxaziridine ring by TP to form TOP as is found in the mass spectrometric analysis of the CARB products. The foregoing not only strongly indicates that the photoreaction of the growth promoters primarily starts with oxaziridine formation but also that these intermediates are quite unstable and reactive. Therefore the possibility was investigated whether photoreactions would entail deterioration of human serum albumin. Isoelectric focusing of HSA is used as a method to investigate whether the protein has been changed. A change of isoelectric point (pI) is considered to be a symptom of deterioration. It was demonstrated that the growth promoters do change the pI of the main 'band' of HSA under UV-A irradiation (Fig. 4). As the results point out, CARB and CYAD upon irradiation deteriorate the HSA main " b a n d " even faster than QUIN and OLAQ (Fig.4b). In this reaction QUIN and OLAQ are completely photodegradated before the HSA main " b a n d " is deteriorated. The photodegradation of these compounds is complete at 15 and 20 s, respectively (Fig. 5). It is assumed that the monoxides of dQUIN and dOLAQ play a role in this reaction. The fact is that these compounds appeared to display photoreactivity towards

93

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360

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Fig. 6. Change of the UV spectrum of h u m a n serum albumin (2 m g / m l ) after UV exposure (150 W / m2, ~,,u = 370 nm) in the presence of quindoxin ( ), olaquindox ( . . . . . . ). carbadox ( - - - - ) and cyadox ( . . . . ) (each 0.1 mg/ml)); irradiation time 400 s, 200 s, 40 s and 30 s, respectively. [ . . . . . ), HSA alone after 5 rain irradiation.

HSA comparable to that of the mother compounds (unpublished data). This may explain the advancing photodeterioration of the protein after QUIN and OLAQ themselves have already been converted. (Mono-N-oxides are still able to form a reactive oxaziridine). It is worth mentioning that CARB and CYAD, just like the known photoallergens QUIN and OLAQ also deteriorate human serum albumin and are even more reactive upon irradiation. Photoreactivity of the growth promoters with HSA was also investigated by comparing the UV spectrum o f HSA from solutions in which the " 4 . 9 " band (isoelectric focusing) had just disappeared (QUIN 400, OLAQ 200, CARB 40 and CYAD 30 s of irradiation) with the UV spectrum of original HSA. To separate HSA from small molecules (unreacted growth promoters, photoproducts, etc.) gel filtration was applied. The efficacy of gel filtration was checked for each compound by submitting part of an irradiated sample to dialysis - - another, more time consuming method for removing small molecules and the rest to gel filtration. Gel filtration appeared to be efficacious as the recorded UV spectrum of a protein solution was equal to that recorded after 48 h of non-equilibrium dialysis. Thus, it was concluded that UV spectra recorded by diode array detector of the HSA 'peak' in the gel filtration chromatogram are spectra from HSA only, (photo)conjugates consisting of irreversibly bound growth promoter derivative molecules to HSA or from deteriorated HSA. Changes in the UV spectrum o f irradiated protein/growth promoter solution after gel filtration, proved reproducible and rather specific for each compound (Fig. 6). Whether a spectral change also indicates irreversible binding of growth promoter derived molecules to HSA is not proven with this investigation. However that imino-N-oxides upon irradiation and their oxaziridines without light, can irreversibly bind to proteins has been shown earlier [1 ll. All four compounds appeared to be reactive with HSA upon UVA-irradiation, irreversibly changing both the UV spectrum of HSA and the pl of HSA's main component. Although CARB and CYAD are more photostabile than QUIN and OLAQ, -

-

94

they proved to be more photoreactive (Fig. 4b). Taking into account clinical data concerning QUIN and OLAQ, and the results of this investigation, CARB and CYAD should be regarded as potential photoallergens. References 1

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4 5 6 7 8 9 10 11 12 13 14 15 16

17 18 19

20

21 22

A. Aumaitre and J.P. Raynaud, Effectiveness of Carbadox at 50 ppm on the performance of the young pig: methodology and influence of the initial weight. Z. Tierphysiol. Tierernahr. Futtermittelk., 40 (1978) 67. K. yon Bronsch, D. Schneider and F. Rigal-Antonelli, Olaquindox-ein neuer Wachstumspromotor in der Tiererniihrung. Z. Tierphysiol. Tiererniihr. Futtermittelk., 36 (1976) 211. I. Herzig, M. Toulova and B. Pisarikova, Effects of the growth stimulator Cyadox on the digestability of neutrients contained in the feed mixture A 1 for feed lot pigs. Biol. Chem. Zivorisne Vyroby Vet., 20 (1984) 235. S. Zaynoun, B.E. Johnson and W. Frain-Bell, The investigation of Quindoxin photosensitivity. Contact Dermatitis, 2 (1976) 343. P . G . Bedello, M. Goitre, D. Cane and G. Roncarolo, Allergic contact dermatitis to Bayo-n-oxI. Contact Dermatitis, 12 (1985) 284. S. Francalanci, M. Gola, S. Giorgini, A. Muccinelli and A. Scrtoli, Occupational photocontact dermatitis from Olaquindox. Contact Dermatitis, 15 (1986) ! 12. K.W. Scott and T.A.J. Dawson, Photo-contact dermatitis arising from the presence of quindoxin in animal feeding stuffs. Br. J. Dermatol., 90 (1974) 543. M.D. Barratt and K.R. Brown, Photochemical binding of photoallergens to human serum albumin; a simple in vitro method for screening potential photoallergens. Toxicol. Lett., 1331 (1984). I. Kochevar, Photoallergic responses to chemicals. Photochem. Photobiol., 30 (1979) 437. J. Epstein, Photoallergy - - A review. Arch. Dermatol., 106 (1972) 741. G.M.J. Beyersbergen van Henegouwen, In vitro and in vivo research on phototoxic xenobiotics: structure-reactivity relationships. Arch. Toxicol. Suppl., 12 (1988) 3. B. Klein and J. Berkowitz, Pyrazines. I. Pyrazine-N-oxides. Preparation and spectral characteristics. J. Am. Chem. SOC., 81 (1959) 5160. H. de Vries, G.M.J. Beyersbergen van Henegouwen, F. Kalloe and M.H.J. Berkhuysen, Phototoxicity of Olaquindox in the rat. Res. Vet. Sci., 48 (1990) 240. M.J. Haddadin and C.H. lssidorides, Photolysis of a quinoxaline-di-N-oxide. Tetrahedron Lett., 8 (1976) 753. A.A. Jarrar and Z.A. Fatafta, Photolysis of some quinoxaline-l,4-dioxides. Tetrahedron, 33 (1977) 2127. C. Kaneko, I. Yokoe, S. Yamada and M. lshikawa, Three-membered ring system with two hetero atoms. VI. Photochemical synthesis of laH-Oxazirino (2,3-a)quinoxaline derivatives and their thermal reactions. Chem. Pharm. Bull., 14 (I 1) (1966) 1316. G.W.H. Cheeseman and E.S.G. TOrzs, Quinoxalines and related compounds. Part VII. Some reactions of quinoxaline N-oxides J. Chem. Soc. (C), (1966) 157. C. Kaneko, S. Yamada, I. Yokoe and M. lshikawa, Structures of the stable photoproducts derived from quinoline l-oxides and quinoxaline I-oxides. Tetrahedron Lett., 20 (1967) 1873. O. Buchardt and J Feeney, Photochemical studies X. On the photolysis of 2,3-diphenylquinoxaline N-oxide to 2,4-diphenylbenz(d)-1,3,5-oxadiazepine. An NMR study. Acta Chem. Scand., 21 (1967) 1399. H. Kawata, K. Kikuchi and H. Kokubun, Studies on the photoreactions of heterocyclic N-dioxides: identification of the oxaziridine intermediate of quinoxaline-l,4-dioxide. J. Photochem., 21 (1983) 343. Y. Hata and M. Watanabe, Is oxygen abstraction by nucleophilic reagents a characteristic reaction of oxaziridines? J. Org. Chem., 46 (1981) 610. L. Homer und E. Jiirgens, Notiz fiber Darstellung und Eigenschaften einiger lsonitrone (Oxazirane) Chem. Ber., 90 (1957) 2184.

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Photochemical reactions of quindoxin, olaquindox, carbadox and cyadox with protein, indicating photoallergic properties.

Quindoxin (quinoxaline-1,4-dioxide), a former 'growth promoter' used in animal husbandry, has been taken from the market because of its photoallergic ...
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