Discussion: session 3. Policy issues in applying epidemiologic evidence.

A MAJOR pathway of steroid metabolism in rat mammary carcinomas is the reduction of testosterone to 5a dihydrotestosterone and 5a androstanediol (King, Gordon and Helfenstein, 1964; Miller, Forrest and Hamilton, 1974). In vitro addition of oestradiol 17/? to incubations of hormone-dependent rat mammary carcinomas is associated with an inhibition of 5a reduction of testosterone (Miller, 1976a). The aim of the present study was to determine if in vivo hormone manipulation was associated with similar changes. A single i.v. injection of 5 mg 7-12dimethylbenzanthracene (DMBA) was given to 24 randomly bred female Sprague Dawley rats at 50 days of age. The size of the tumours which were induced was monitored twice weekly by measuring with calipers 2 diameters at right angles. When the tumours were 2 x 2 cm in size, animals were allocated to one of 3 groups. Those in Group I were killed without further treatment, those in Group II were oophorectomized and killed 14 days later and those in Group III were oophorectomized but 14 days later received daily s.c. injections of 1 jug oestradiol in corn oil for a further 14 days, when they were killed. In all animals, oophorectomy led to regression oftumours; all those subsequently given oestradiol showed regrowth. Tumours were harvested at death and treated at 0°C. Each was finely sliced in Krebs Ringer phosphate buffer pH 7-4 (10 ml/g tumour) and an NADPHgenerating system (200 ,umol glucose-6Accepted 25 May 1976

A MAJOR pathway of steroid metabolism in rat mammary carcinomas is the reduction of testosterone to 5a dihydrotestosterone and 5a androstanediol (King, Gordon and Helfenstein, 1964;Miller, Forrest and Hamilton, 1974). In vitro addition of oestradiol 17/? to incubations of hormone-dependent rat mammary carcinomas is associated with an inhibition of 5a reduction of testosterone (Miller, 1976a). The aim of the present study was to determine if in vivo hormone manipulation was associated with similar changes.
A single i.v. injection of 5 mg 7-12dimethylbenzanthracene (DMBA) was given to 24 randomly bred female Sprague Dawley rats at 50 days of age. The size of the tumours which were induced was monitored twice weekly by measuring with calipers 2 diameters at right angles. When the tumours were 2 x 2 cm in size, animals were allocated to one of 3 groups. Those in Group I were killed without further treatment, those in Group II were oophorectomized and killed 14 days later and those in Group III were oophorectomized but 14 days later received daily s.c. injections of 1 jug oestradiol in corn oil for a further 14 days, when they were killed. In all animals, oophorectomy led to regression oftumours; all those subsequently given oestradiol showed regrowth.
Tumours were harvested at death and treated at 0°C. Each was finely sliced in Krebs Ringer phosphate buffer pH 7-4 (10 ml/g tumour) and an NADPHgenerating system (200 ,umol glucose-6-  (Miller et al., 1974). The metabolism of testosterone by conversion to 5ac dihydrotestosterone and 5ca androstanediol were determined by measuring the percentage incorporation of radioactive label into the appropriate metabolites after correction for recovery losses. The DNA content of the tumours was determined by a modification of the method of Burton (1956).
Although 5a dihydrotestosterone was a metabolite of testosterone in all tumours, there was no significant difference in its production between the animal groups, despite a lowered mean production in the group receiving oestrogen. In contrast, the conversion of testosterone to its other major 5ac reduced product, 5a androstanediol, was clearly influenced by hormone manipulation, and was significantly increased in tumours from oophorectomized rats. The subsequent administration of oestradiol was associated with a significant decrease in production of 5cx androstanediol, to levels comparable with tumours from the intact group.
Sufficient material was available for determination of DNA content in 6 tumours from each group. No significant differences were observed (Table II). These results indicate that, in female Sprague Dawley rats bearing DMBAinduced mammary carcinomas, oophorectomy is associated with an increase in tumour metabolism of testosterone, a phenomenon which may be reversed by in vivo administration of oestradiol. A similar pattern was observed in the conversion of testosterone to 5ac androstanediol. Increased production of 5a androstanediol in tumours from oophorectomized animals alone would account for the higher levels of testosterone metabolized by these tumours. In contrast, reduced formation of 5ac androstanediol does not fully account for the decreased levels of testosterone metabolized in tumours from oestrogentreated animals: reduced conversion to 5ac dihydrotestosterone in these tumours also contributes to the decreased metabolism of testosterone.
As the DNA content of tumours from each animal group was similar, these changes are unlikely to be caused by differences in tumour cellularity. Furthermore, the decreased metabolism and conversion of testosterone to 5ac androstanediol following in vivo administration of oestrogen may be reproduced in vitro by addition of oestradiol to incubations of hormone-dependent rat mammary carcinomas (Miller, 1976a).
Because the methods used in these studies estimate the total production of all 4 isomers of 5ax androstanediol (Miller, 1976a), it is not possible to determine if the production of a particular isomer is preferentially affected by hormone manipulation.
However, only 17,? isomers of androstanediol were identified as metabolites of testosterone in human breast cancer (Cameron et al., 1971).
It is possible that oophorectomy and oestrogen administration affect tumour metabolism indirectly, by respectively lowering and raising circulating levels of prolactin, to which the growth of DMBAinduced tumours are particularly sensitive (Meites, 1972;Pearson et al., 1972). Nevertheless the in vitro addition of oestradiol (Miller, 1976a), as well as prolactin (Miller, 1976b), has been shown to influence testosterone metabolism by DMBA rat tumours and both hormones may therefore be implicated in the changes effected by the endocrine manipulations described in this study.
Whether the effects of oestrogen administration are caused directly by oestradiol 17/l, or indirectly by prolactin secretion, or both, the lower synthesis of 5a dihydrotestosterone and 5a androstanediol in growing tumours from animals given oestrogen, as compared with regressing tumours after oophorectomy, is in keeping with the growthinhibiting properties of 5a reduced steroids (Huggins and Mainzer, 1957). However, without further work to determine the sequence of events following hormone manipulation, it is not possible to indicate whether changes in steroid metabolism occur before or concurrently with those in tumour growth, or whether the change in tumour growth itself leads to differences in metabolism of testosterone.