SHORT COMMUNICATION J . vet. Phunnacol. Thcrap. 10, 172-174, 1987.

Comparative acetylation and hydroxylation of sulfamethoxazole and sulfatroxazole in the snail Cepaea hortensis T. B. VREE*, MONIKA L. VREEt & J. F. M. NOUWSS *Department of Clinical Pharmacy, Sint Radboud Hospital, University of Nijmegen, tDotulabs, Weezenhof 3508, Nijmegen and SRVV kring 6, Wolfskuilseweg 279, Nijmegen, The Netherlands

Acetylation is a common conjugation (phase 11) reaction in the mammalian metabolism of sulfonamides. In addition to acetylation, hydroxylation and glucuronidation of the hydroxy metabolites also take place in mam- mals. Although both reaction pathways seem to be competitive, each depends upon the molecular structure of the sulfonamide and the enzymes present in the species (Vree et al., 1985). It has recently been shown that the snail Cepaea hortmis is able to hydroxylate and acetylate sulfonamides, and that for sulfa- dimidine, hydroxylation is a more important pathway than acetylation (Vree & Vree, 1983; Vree et al., 19866). The acetylation and hydroxylation pathways have therefore ex- isted for a period of at least 600 million years (Solem, 1974). Sulfatroxazole is mainly hyd- roxylated (70%) in man, conjugation by acetylation accounting for only 15% of the administered dose (Vree et al., 1986~). Sul- famethoxazole, however, is 70% acetylated and 30% hydroxylated (Vree et al., 1985). It has been demonstrated that snails are able to acetylate sulfamethoxazole (Vree et al., 1983), but hydroxylation of this compound has not yet been demonstrated. Sulfamethoxazole and sulfatroxazole are structural analogues. The extra methyl group at the 4-position in sulfatroxazole favours metabolism in man by the hydroxylation pathway, rather than the acetylation pathway. The aim of this study was to investigate whether the snail is able to

Correspondence: Dr T. B. Vree, Department of Clinical Pharmacy, Sint Radboud Hospital, Univer- sity of Nijmegen, Geert Grooteplein Zuid 8, Nijmegen, The Netherlands.

hydroxylate sulfatroxazole and sulfamethox- azole like man. It may be demonstrated that the rate of hydroxylation of the sulfonamide is governed by its molecular structure.

One small tank contained 21 snails Cepaea hwtensis (120 g total body weight). In another small tank, 19 snails Cepaea hortensis (85 g total body weight) were placed. To the tank with 21 snails, 44.4 mg sulfatroxazole in 55 ml water were added, and to the tank with 19 snails, 41.3 mg sulfamethoxazole in 55 ml water were added. Sulfatroxazole was obtained from Uvens Kemiske Fabrik (Copenhagen, Den- mark) and sulfamethoxazole was obtained from Hoffman LaRoche (Mijdrecht, The Netherlands). The snails moved normally within their tanks, maintaining their feeding activity and swallowing water containing the sulfonamide. This was probably excreted by the kidney, in the mucus and in the faeces, and re-dissolved in the water. The glass walls of the tanks were rinsed with the solution in order to collect all mucus, faeces and urine. After each rinsing procedure, an aliquot of 2 ml was taken and stored at -20°C pending analysis. For each 2 ml sample removed, 2 ml of water was added in order to keep the water content of the tanks as constant as possible. Sample collection took place over a period of 60 h. Sulfatroxazole (S) with its metabolites 5-hydroxy-sulfatroxazole (SOH) and N4-acetylsulfatroxazole (N4), and sulfamethoxazole (S) with its metabolites 5-hydroxy- (SOH) and N4-acetylsulfameth- oxazole (N4) were analysed by high perform- ance liquid chromatography (HPLC, Vree el al., 1985, 1986~). Deglucuronidation of the

172

Acetylation and hydroxylation of sulfonamides 173

water samples was carried out as previously described (Nouws et al., 1985a.6).

Figure la shows the concentration-time curves of sulfatroxazole and its three metabo- lites for 21 snails. Hydroxy- and acetyl- metabolites of sulfatroxazole are rapidly formed and the percentage of the dose cumulatively excreted in the water increases to 10.7% for 5-hydroxysulfatroxazole (8.2% SOH, and 2.5% SOHgluc.), and to 0.8% for N4-acetylsulfatroxazole.

Figure 1 b shows the concentration-time curves of sulfamethoxazole and its two metabolites for 19 snails. The hydroxy- and acetyl-metabolites appear rapidly, and the percentage of the dose cumulatively excreted increases to 3.5% for 5-hydroxysulfa- methoxazole (SOH), and to 4.0% for N4-acetylsulfamethoxazole. No glucuronide of 5-hydroxysulfamethoxazole could be de- tected.

Sixty per cent of the mass balance was unaccountr ' for in both experiments. The initial concentration at t = 0 represents 50% of the administered dose. A similar figure was found for sulfadimidine in snails (Vree et al., 19866). It is possible that the sulfonamide is adsorbed onto the glass wall of the tanks, or adsorbed onto the shell, or absorbed by the skin of the animals. In conclusion, this experi- ment demonstrated that sulfatroxazole was mainly hydroxylated in the snail Cepaea hmten- sts, but sulfamethoxazole was acetylated and hydroxylated at similar low rates. Hydroxyl- ation at the 5-methyl position is facilitated by the methyl group at the 4-position in sulfa- troxazole in many species (Vree et al., 1986a; Haenen et al., 1986).

REFERENCES

Haenen, O.L.M., Grondel, J.L. & Nouws, J.F.M. (1986) Pharmacokinetics of oxytetracycline, sul- phatroxazole and sulphadimidine in the carp. In Comparative Veterinary Phannacology, Toxicology and Therapy. MTP Press, Lancaster.

Nouws, J.F.M., Vree, T.B., Baakman, M., Dries- sens, F., Smulders, A. & Holtkamp, J. (1985a) Disposition of sulfadimidine and its N4-acetyl and -hydroxy metabolites in horse plasma. J o u m l of Veterinary Pharmacology and Therapeutics, 8, 303- 311.

Nouws, J.F.M., Vree, T.B., Breukink, H.J., Baak- man, M., Driessens, F. & Smulders, A. (19856)

I I I I I I 1 1 0 10 20 30 LO 50 60

1 d ?O 10 :O 20 20 60

Time (h)

FIG. 1 . (a) Cumulative excretion of 5-hydroxy- sulfatroxazole (SOH), its glucuronide (SOHgluc.), and N,-acetylsulfatroxazole (N4) by 2 1 snails Cepaea hortemis after addition of 44.4 mg sulfatroxazole to the tank. (b) Cumulative excretion of 5- hydroxysulfamethoxazole (SOH) and Nq- acetylsulfamethoxazole (N4) by 19 snails Cepaea hortnrris after addition of 41.3 mg sulfamethoxazole to the tank.

Dose dependent disposition of sulphadimidine and of its N4-acetyl and -hydroxy metabolites in plasma and milk of dairy cows. The Vetminary

Solem, W . (1974) The Shell Makers: Introducing Molluth. Wiley Interscience Publications, New York.

Vree, T.B., & Vree, M.L. (1983) Acetylation of

QUUT&L'Y, 7. 177-186.

174 T. B . Vree, M . L. Vree k3 J . F. M. Nouws

sulphamethoxazole by the snail Cepaea hortensis. kinetics, metabolism and renal excretion of sulfa- Journul of Vetm'nuy Phurmacohgy and Therapeutics, troxazole and its 5-hydroxy- and N,-acetyl- 7, 239-241. metabolites in man. Biophannaceutics and Drug

Dirpositbn, 7, 239-252. Metabolism of sulfonamides. Antibiotics and Vree, T.B., Vree, M.L. & Nouws, J.F.M. (19866) Chemotherapy, 34, 5-65. Acetylation and hydroxylation of sulphadimidine

Vree, T.B., Hekster, Y.A., Tijhuis, M.W., Ter- in the snail Cejma hurtensir. Zmtralbkztt fur Veler- mond, E.F.S. & Nouws, J.F.M. (1986a) Pharmaco- in& Medizin, 3, 633-636.

Vree, T.B., Hekster, Y.A. & Tijhuis, M.W. (1985)