J. Agric. Food Chem., Vol. 26, No. 1, 1978
Estrogenic Activity of o,p’-DDT Metabolites
140
Estrogenic Activity of o,p’-DDT Metabolites and Related Compounds Joel Bitman,* Helene C. Cecil, Susan J. Harris, and Vernon J. Feil
Rat uterine glycogen responses were determined for 20 metabolites or compounds related to metabolites of o,p’-DDT to determine relationships of structure to estrogenic activity. Methoxy analogues of o,p’-DDA, o,p’-DDD, and o,p’-DDE and a glycine conjugate of o,p’-DDA exhibited only minimal estrogenic activity. Esterification of o,p’-DDA reduced this activity as did conjugation with serine. The presence of two methoxy groups rendered the o,p’-DDA completely inactive. Only o,p’-DDT analogues were active, demonstrating that a stable ethane chain was necessary. Activity was retained in the o,p’-DDT series if an hydroxy or methoxy group was present in the 3 or 4 position of the o-chloro ring. Estrogenic activity in decreasing order was: 3-hydroxy- > unsubstituted N 4-methoxy- N 5-hydroxy- > 5-methoxy-o,p’-DDT.
The estrogenic activity of commercial DDT has been demonstrated to be due to the presence of the ortho,para’ isomer of DDT (Welch et al., 1969, Bitman, et al., 1968). The geometric similarity of the DDT molecule to the synthetic estrogen, stilbestrol, led us to examine DDT and 52 analogues, homologues, and structurally related compounds in an attempt to determine relationships of structure to estrogenic activity (Bitman and Cecil, 1970). We postulated that active estrogens would be derived from o,p’-DDT by metabolism of the ring bearing the orthochlorine atom and would be phenolic metabolites. Whereas the metabolism of p,p’-DDT has been investigated in many species (Menzie, 1969; Matsumura, 1975), the metabolism of o,p ’-DDT has received little attention. Recently, the metabolism of o,p’-DDT was studied in rats (Feil et al., 1973) and in chickens (Feil et al., 1975). After the administration of 14C-labeled o,p’-DDT to rats, 13 compounds were identified in rat feces. After dosing with o,p’-DDT-I4C, 16 compounds were identified in chicken excreta. These studies demonstrated that the ortho chloro ring was extensively metabolized, whereas the para chloro ring remained intact (Feil et al., 1975). Eight of the 13 metabolites in rats and 13 of the 16 metabolites in chickens were hydroxy and methoxy compounds. Since certain of these compounds fit the theoretical requirements we had formulated for estrogenicity, we decided to test several of these metabolites to confirm relationships of structure to estrogenic activity. Several closely related analogues, also prepared synthetically, were tested because they may be metabolites at low concentrations even though they were not isolated as metabolites. METHODS
We used the sensitive 18-h glycogen response of the rat uterus as a measure of estrogenic activty (Bitman et al., 1965; Cecil et al., 1971). The potency of active compounds is reported in terms of the minimal subcutaneous dose which will increase glycogen to a level significantly different from control. Test substances were dissolved in olive oil or an aqueous ethanol solution and injected subcutaneously into groups of six-eight rats at a screening dose rate of 8 mg/rat. Immature female Wistar rats (21-23 days old; 36-48 g) were killed 18 h after the injection; uteri were quickly excised, weighed, and analyzed for glycogen by the anthrone procedure (Seifter et al., 1950). Substances showing activity at the 8-mg screening level were successively tested at lower dosages, 4, 2, 1, etc. to 0.10 mg. Agricultural Research Service, US. Department of Agriculture, Beltsville, Maryland 20705 and Fargo, North Dakota 58102.
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