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Uptake of dehydroepiandrosterone sulphate into human breast cyst fluids
The Breast (1997) 6. 12-16 0 1997 Pearson Professional Ltd
ORIGINAL ARTICLE
Uptake of dehydroepiandrosterone sulphate into human breast cyst fluids J. M. Dixon, J. Telford*, R. A. Elton+ and W. R. Miller* University Department of Surgery, Royal Infirmary, VCRF Department of Medical Oncology, Western General Hospital, “Medical Statistics Unit, University of Edinburgh Medical School, Edinburgh, UK S U M M A R Y. 7-alpha tritiated dehydroepiandrosterone (DHA) sulphate was infused over a 24-h period to five patients with multiple breast cysts and over 48 h to 11 patients. Cysts were aspirated and blood taken at various times during the infusion and during a 2-year follow-up period. Plasma levels of radioactivity remained constant during the course of infusion whereas radioactivity in cyst fluids increased with time of infusion and by 20 h levels of radioactivity in cyst fluid were frequently greatly in excess of those in plasma. This active uptake of radioactivity was evident in four of five patients infused for 24 h and in all 11 patients studied over 48 h. Not all cyst fluids demonstrated active uptake. This was only seen in Type I cysts (apocrine - identified by electrolyte composition) and not in Type II cysts. Cysts were aspirated from nine patients during the 2-year follow-up period. All cysts contained levels of radioactivity beyond that detected in plasma (which was negligible) and in three patients levels of radioactivity from cysts aspirated from follow-up were higher than the levels found in cyst fluids aspirated during the period of infusion. Even at 2 years after infusion detectable levels of radioactivity were present in breast cysts fluids. Characterization of the radioactivity showed that it was associated with DHA-sulphate. This study has demonstrated active uptake of DHA-sulphate into Type I cyst fluids. Although the clinical significance remains to be determined, it demonstrates that breast epithelium may actively concentrate and retain substances from plasma. This means that breast epithelium may be exposed to high concentrations of xeno-biologicals for prolonged periods.
INTRODUCTION
concentrations are achieved, although a small study in which two patients were given radioactively labelled DHAsulphate did show accumulation of radioactivity in breast cyst fluids.’ The time course of this uptake and the differential uptake between the two types of cysts were not examined. The aim of the present study was to investigate more fully the uptake of DHA-sulphate into human breast cyst fluids.
Approximately 7% of all women in the Western world present to hospital with a clinically palpable breast cyst.’ The total number of cysts in individuals varies greatly. Approximately half of all patients develop a single cyst, one-third have from two to five cysts and the remainder develop more than five.’ Two populations of human breast cysts can be defined on the basis of their cyst fluid electrolyte composition and biochemistry. Type I cysts have a [sodium]/[potassium] ratio [Na+]/[K’]c3 and Type II cysts have a [Na’]/[K+]>3.2,3 The two populations are lined by different epithelium, Type I cysts being lined by apocrine and Type II cysts by flattened epithelium.4 These two cyst types also differ in the amounts of steroid hormones present within their cyst fluid.* For example, the mean concentration of the conjugated steroid, DHA-sulphate, found in Type I cysts is over loo-times greater than that in Type II cysts, which contain similar levels to those found in plasma? Little is known about the mechanism by which these Address Western
correspondence fo: J. M. Dixon, Edinburgh General Hospital, Crewe Road, Edinburgh
PATIENTS, MATERIALS
AND METHODS
Patients presenting to the Edinburgh Breast Unit with multiple palpable breast cysts were entered into the study. None were receiving medication for their condition and none were taking diuretics. Ethical permission for the investigation was obtained and the nature of the study explained to each patient before obtaining written consent. Over either 24 h (5 patients) or 48 h (11 patients), 1 MBq of 7-alpha tritiated (3H) DHA-sulphate was infused. Over the first hour, 10 ml of infusion was given and the remainder infused at a constant rate using an infusion pump. Blood and cyst fluids were obtained by venopuncture and needle
Breast Unit, EH4 2XU, UK
12
Uptake of dehydroepiandrosterone sulphate into human breast cyst fluids aspiration at varying time points during the period of infusion, while patients were sedated with intravenous Midazolam. In 15 patients the cyst fluids were aspirated during the period of infusion but in one patient who was infused over 48 h the cyst fluids were aspirated 7 days after the start of infusion. Blood was centrifuged at 2500 rpm for 10 min and the resultant plasma taken and frozen at -20°C. Cyst fluids were immediately stored at -20°C until batch analysis. The radioactivity in plasma and cyst fluids was determined by counting 25 ~1 and 250 ~1 aliquots in duplicate with and without the addition of 2000 dpm [3H] steroid (to monitor internal quenching) in 10 ml of NE260 scintillant. To confirm that radioactivity was associated with DHA-sulphate samples were extracted with ether and the aqueous phase subjected to acid solvolysis. The solvolysed material was extracted into ethyl acetate and run on thin layer chromatography together with authentic steroid standards. Electrolyte composition of cyst fluid was determined by flame photometry and cysts were classified into Type 1 if cyst fluid had a sodium:potassium ratio [Na’/K’] of <3 and were classified into Type II if the ratio was 23.‘S2,4 To compare uptake with sodium:potassium ratio, data from three patients infused over 48 h who had large numbers of cysts aspirated at 48-h time point were correlated. As the data varied over several orders of magnitude, the correlation was between the log of the ratio of sodium: potassium and the log of the radioactivity.
RESULTS Infusion over 24 h Representative results from two of the five patients infused are shown in Figure 1. Levels of radioactivity in plasma increased during the first few hours of infusion following the bolus but then remained constant so that between 20 and 24 h there was tight range of values for all five patients (3200-4700 dpm/ml). In contrast levels of radioactivity in cyst fluids tended to be low during the initial stages of infusion and in no patient was there more radioactivity in cysts fluids than in plasma during this early period. Cysts fluids aspirated at 20 h and beyond frequently contained high levels of radioactivity (Fig. 1). In certain patients levels of radioactivity were greater than those ever seen in plasma. A total of 60 cysts were aspirated from these five patients; 45 were Type I and 15 were Type II. Of these 66, 23 cysts contained greater levels of radioactivity than in plasma and all 23 were Type I cysts. There were 30 cysts aspirated at the 24-h time point and 17 of these contained higher levels of radioactivity than the corresponding level in plasma (Table 1). All fluids with excess radioactivity were Type I
13
Thousands 14 m
n
Type I I cysts
m
Type I cysts<
a dpmlml 6
2h
4h
A
6h
20h
24h
Time Thousands
15
dpmlml 10
/ B
-Type
Fig. 1 varying (B) Cyst infusion
II cysts
~Plasma
m
Type I cysts
(A) Levels of radioactivity in cyst fluids and plasma aspirated at times during a 24-h infusion (patient number 1 in Table 1). fluids and plasma aspirated at 24 h in a patient having a 24-h of radioactive DHA-sulphate (patient number 4 in Table 1).
cysts and none of the six Type II fluids aspirated at 24 h contained substantial amounts of radioactivity. Although four of the five women had cysts which contained more radioactivity than plasma, one patient (patient five) never accumulated more radioactivity in cyst fluid than in plasma.
Infusion over 48 h Representative results from two patients are shown in Figure 2. Levels of radioactivity in plasma were remarkably constant between 24 and 48 h and there was a narrow range between patients (1600-3500 dpm/ml). Levels of radio-
Table levels Patient
1 Proportion of cyst fluids containing in plasma as sampled at 24 h Type 1 fluids
Type II fluids
Excess
Excess
Total
Total
radioactivity
in excess Total Excess
Total
1 2 3 4 5
6 3 3 5 0
I 3 5 1 2
0 0 0 0 0
1 0 0 3 2
6 3 3 5 0
8 3 5 IO 4
Total
17
24
0
6
17
30
of
14
The Breast Table levels
Thousands 14 m
Plasma
Patient
6 6
0
24h
46h
Time
A
radioactivity
in excess
of
hsI Type I cysts
12 n
dpmlml
2 Proportion of cyst fluids containing in plasma as sampled at 48 h Type I fluids
Type II fluids
Total
Excess
Total
Excess
Total
Excess
Total
1 2 3 4 5 6 7 8 9 10
9 10 7 2 7 8 5 5 6 8
9 13 10 5 8 9 5 8 9 13
0 0 0 0 0 0 0 0 0 0
0 6 1 1 0 2 0 0 0 4
9 10 7 2 7 8 5 5 6 8
9 19 11 5 8 11 5 8 9 17
Total
67
89
0
14
67
103
14 12 10
dpm/ml
a 6 4 2 0
B
I
Type II cysts
~Plasma
6sI Type I cysts
Fig. 2 (A) Levels of radioactivity in cyst fluids and plasma at 24 and 48 h from a patient infused with radioactive DHA-sulphate over 48 h (patient number 1 in Table 2). (B) Levels of radioactivity in cyst fluids and plasma at 48 h in a patient having a 48-h infusion of radioactive DHA-sulphate (patient number 2 in Table 2).
activity in cyst fluids varied enormously from undetectable levels to in excess of 32 000 dpm/ml. The amount of radioactivity in cyst fluids tended to increase from 24 to 48 h. At 48 h the majority of cyst fluids contained more radioactivity than the corresponding level of plasma with some cyst fluids containing as much as 15fold greater excess than plasma. A total of 145 cysts were aspirated from the 10 patients whose cysts were aspirated during the 48-h period of infusion. Of these, 117 were Type I and 28 were Type II. Eighty-six cysts contained more radioactivity than the corresponding level in plasma and all 86 were Type I cysts. All patients by 48 h had at least some cysts which contained more radioactivity than the corresponding level in plasma (Table 2). Of a total of 103 cyst fluids aspirated at 48 h, 67 had levels of radioactivity higher than plasma (Table 2). All 67 were Type I and none of the 14 Type II cyst fluids accumulated radioactivity beyond that in plasma (Table 2).
Forty-eight-hour
infusion: cysts aspirated at 7 days
A total of five cysts were aspirated from this patient, three were Type I and two Type II (Table 3). The plasma level of
radioactivity at 7 days was low. Both Type II cysts aspirated had levels of radioactivity at 7 days which were in excess of those in plasma but lower than the circulating levels of radioactivity observed in other patients during the course of infusion. The three Type I cysts aspirated at 7 days contained much higher levels of radioactivity with a relative excess varying from 20 to 75-times the level in plasma at 7 days (Table 3). Cysts aspirated during follow-up During the 26-month period following the infusion with 7alpha tritiated DHA-sulphate a total of 39 cysts were aspirated from nine of the patients studied. The interval between infusion and aspiration varied between 3 weeks and 26 months. By 3 weeks the amount of radioactivity in plasma was negligible, but radioactive label could be detected in cyst fluids in samples taken over 2 years after infusion. Levels of radioactivity in Type I cyst fluids were much higher than those in Type II fluids. The levels of radioactivity in the 31 Type I cysts are plotted in Figure 3. Two patients were interesting in that the radioactivity found in cyst fluids aspirated 3 weeks and 3 months respectively after infusion were in excess of those in cyst fluids aspirated during the course of infusion.
Table 3 infusion
Radioactivity Breast
Plasma at 7 days cyst fluid
in cyst fluids
Type
and plasma
dpm
7 days after 48-h Relative
360 L L L R R
II II I I I
1440 960 26 960 8240 8120
4.0 2.6 74.9 22.9 22.6
excess
Uptake of dehydroepiandrosterone
sulphate into human breast cyst fluids
15
3 3 Ratio H in cyst fluid I H in plamla during infusion
. I.0
.
.
l
’
0.5
. l . . .
.‘, 0. 0.
. . . .
. . .
:: .*
Patient A3 .2 5
Fig. 3 Levels of radioactivity in breast cyst fluids aspirated intervals following infusion of radioactive DHA-sulphate.
Correlation between radioactivity with sodium:potassium ratio
01
at various Fig. 4 Correlation of log of radioactivity ratio. Data from three patients, analysing
with log of sodium:potassium only cysts aspirated at 48 h.
at 48-h compared
Data from three patients who had 43 cysts aspirated at 48 h after radioactive DHA-sulphate infusion showed that there was a direct correlation between the log of the sodium: potassium ratio and the log of the radioactivity (Fig. 4). This correlation was significant at FYO.001. In fact, there was a very clear inverse relationship and the majority of the points were close to a fitted 45” line with the exception of three cysts from a single patient.
Biochemical studies The tritium remained in the aqueous fraction after ether extraction (i.e. it was conjugated) and on acid solvolysis was extractable into an organic solvent. This extracted material had a similar chromatographic mobility to an authentic DHA standard on thin layer chromatography. This was true for cyst fluids taken during and after infusion and was irrespective of cyst type.
DISCUSSION The present observations demonstrate that 7-alpha tritiated DHA-sulphate appears in cyst fluids within hours of intravenous administration and that by 24 h the levels of radioactive DHA-sulphate in cyst fluids may exceed those in plasma. This active uptake appears to be specifically associated with Type I (apocrine) cysts and follow-up data suggest that the DHA-sulphate is retained within breast fluids over a prolonged period. The only other report of uptake of radioactive steroids into breast fluids was a limited study of two patients who had a bolus injection of labelled DHAsulphate following which one cyst was aspirated at three separate time points over a 48-h period.’ No details were subsequently given of the types of cysts aspirated or the
relative accumulation of radioactivity in cysts as compared to levels in plasma. By using patients with multiple cysts and aspirating fluids at different times during a constant intravenous infusion of radioactively labelled steroid it has been possible to study the extent and time course of the uptake of radioactively labelled DHA-sulphate into breast cysts. While the data demonstrate that there were differences between cysts in individual patients and that there was some variation from patient to patient, there was a general consistency of results which allows certain conclusions to be drawn. First, while levels of radioactively labelled DHAsulphate remained constant during the period of infusion, those in cyst fluids increased with time. Although radioactivity was detected in cyst fluids as early as 2 h, the levels were low and never exceeded those in plasma. In contrast, cysts aspirated at later time points frequently contained levels of radioactivity above those found in’ plasma and some cysts contained as much as 15times more radioactivity than in plasma. By the end of the infusion four of the five patients infused over 24 h and all the patients infused over 48 h had cysts which contained more radioactivity than plasma. The amounts of radioactive label in cyst fluids aspirated at 48 h were generally higher than those observed during the 24-h infusion. The further observation that the single patient who had a cyst aspiration delayed until one week after the completion of a 48-h infusion displayed among the highest levels of radioactivity in cyst fluids suggests that there is prolonged and persistent uptake of [3H] DHA-sulphate from the circulation. Although the majority of cysts accumulated [‘HI DHAsulphate in levels greater than that found in the circulation, a substantial number did not. When cysts were classified into Types I and II based on their electrolyte composition, only Type I cysts accumulated greater levels of radioactivity than plasma. The reason why some but not all Type I cysts accumulated radioactivity is uncertain but we were
16
The Breast
Table
4
Patient
Radioactivity Maximum levels plasma
in recurrent
cysts
Time of sample (h) cyst (h)
1
6880 (6)
11 960 (24)
4 5 2
4800 (6) 4760 (24) 2480 (48)
16 760 (24) 3800 (24) 20 280 (48)
6 7
2640 (48) 2960 (48)
12 120 (48) 11 240 (48)
8
3360 (48)
4280 (48)
10
3320 (48)
7400 (48)
11
2640 (48)
20 280 (48)
Recurrent sample
Type
Time of aspiration
590 530 510 1560 1840 1040 1720 400 185 120 5480 160 10040 1480 3160
I I I II I I I II II Plasma I Plasma I I I
18 months 18 months 18 months 3 months 3 months 3 months 3 months 3 weeks 3 weeks 3 weeks 3 weeks 3 weeks 3 months 6 months 18 months
unable to determine any other factor which was influential. The uptake of radioactive DHA-sulphate into Type I, but not Type II cysts, is compatible with our previous studies13294 which have demonstrated that endogenous levels of DHAsulphate in Type I cysts are on average loo-fold higher than those in the circulation, whereas Type II cysts show levels similar to those in plasma. It had previously been a matter of conjecture as to whether these high levels were due to in situ synthesis or derivation from plasma. The present study suggests that active uptake from plasma could account for these high levels. The clinical relevance of DHA-sulphate in cyst fluids has yet to be determined. For a hormone, DHA-sulphate is produced in large quantities6 and has relatively little biological activity unless metabolised.’ However, although an androgen in structure, at high concentrations it can behave as an oestrogen and induce oestrogenic responses.* DHA and its reduced metabolite, delta-5androstenediol can maintain the growth of oestrogen-dependent breast cancer cell lines.9~‘0It is thus of interest that the patients with Type I cysts have been shown to have an increased risk of breast cancer, as have women with apocrine metaplasia within their breast.” Furthermore, the accumulation of DHAsulphate may be a marker for other more active agents which could be accumulated by the same mechanism. For example, a recent report has described the accumulation of bile acids in breast cyst fluids.12 These observations indicate
the potential for breast epithelium to concentrate and retain xenobiologicals. Such prolonged exposure to noxious compounds could potentially explain the increased cancer risk which has been associated with Type I breast cystic disease.‘33’4
References 1. Dixon J M. Cystic disease of the breast. In: Smallwood, Taylor, eds. Benign breast disease. London: Edward Arnold, 1990; 66-84. 2. Miller W R, Dixon J M, Scott W N, Forrest A P M. Classification of human breast cysts according to electrolyte and androgen composition. Clin Oncol 1983; 9: 227-232. F D, Schwartz M K, Fleisher M. Cation 3. Bradlow H L, Skidmore levels in human breast cyst fluids. Clin Oncol 1981; 7: 388-390. 4. Dixon J M, Miller W R, Scott W N, Forrest A P M. The morphological basis of human breast populations. Br J Surg 1983; 70: 604-606. 5. Miller W R, Dixon J M, Forrest A P M. Hormonal correlates of apocrine secretion in the breast. Ann N Y Acad Sci 1986; 464: 275-287. 6. Abraham G E. Ovarian and adrenal contribution to peripheral antigens during the menstrual cycle. J Clin Endocrinol Met 1974; 39: 340-346. C, Dray F et al. An adrenal-secreted 7. Baulieu E E, Corpechot ‘androgen’; dehydroandrosterone sulphate, its metabolism and a tentative generalization of the metabolism of other steroid conjugates in man. Ret Prog Horm Res 1965; 21: 411-500. 8. Adams J, Garcia M, Rochefort H. Estrogenic effects of physiological concentrations of 5-androstene-3P, 17P-diol and its metabolism in MCF, human breast cancer cells. Cancer Res 1981; 41: 4720-4725. 9. Boccuzzi G, Brignardello E, Di Monaco M, Forte C, Leonardi I, Pizzini A. Influence of dehydroepiandrosterone and 5-en-androstene3-beta, 17 beta-diol on the growth of MCF-7 human breast-cancer cells induced by 17-beta-estradiol. Anticancer Res 1992; 12: 799-803. 10. Hackenberg R, Turgetto I, Filmer A, Schulz K-D. Estrogen and androgen receptor-mediated stimulation and inhibition of proliferation by androst-5-ene-3-beta, 17-beta-diol in human mammary cancer cells. J Steroid Biochem Molec Biol 1993; 46: 597-603. 11. Schuerch C, Rosen P P, Hirota T et al. A pathologic study of benign breast diseases in Tokyo and New York. Cancer 1982; 50: 1899-1903. 12. Javitt N B, Budai K, Miller D G, Cahan A C, Rasu N, Levitz M. Breast gut connection - origin of chenodeoxycholic acid in breast cyst fluid. Lancet 1994; 343: 633-635. 13. Miller W R, Scott W N, Harris W H, Wang D. Using biological measurements can patients with benign breast disease who are at high risk for breast cancer be identified. Cancer Det Prev 1992; 16: 99-106. 14. Angeli A, Dogliotti L, Naldoni C et al. Steroid biochemistry and categorization of breast cyst fluid - relation to breast cancer risk. J Steroid Biochem Mol Biol 1994; 49: 333-339.
ORIGINAL ARTICLE
Uptake of dehydroepiandrosterone sulphate into human breast cyst fluids J. M. Dixon, J. Telford*, R. A. Elton+ and W. R. Miller* University Department of Surgery, Royal Infirmary, VCRF Department of Medical Oncology, Western General Hospital, “Medical Statistics Unit, University of Edinburgh Medical School, Edinburgh, UK S U M M A R Y. 7-alpha tritiated dehydroepiandrosterone (DHA) sulphate was infused over a 24-h period to five patients with multiple breast cysts and over 48 h to 11 patients. Cysts were aspirated and blood taken at various times during the infusion and during a 2-year follow-up period. Plasma levels of radioactivity remained constant during the course of infusion whereas radioactivity in cyst fluids increased with time of infusion and by 20 h levels of radioactivity in cyst fluid were frequently greatly in excess of those in plasma. This active uptake of radioactivity was evident in four of five patients infused for 24 h and in all 11 patients studied over 48 h. Not all cyst fluids demonstrated active uptake. This was only seen in Type I cysts (apocrine - identified by electrolyte composition) and not in Type II cysts. Cysts were aspirated from nine patients during the 2-year follow-up period. All cysts contained levels of radioactivity beyond that detected in plasma (which was negligible) and in three patients levels of radioactivity from cysts aspirated from follow-up were higher than the levels found in cyst fluids aspirated during the period of infusion. Even at 2 years after infusion detectable levels of radioactivity were present in breast cysts fluids. Characterization of the radioactivity showed that it was associated with DHA-sulphate. This study has demonstrated active uptake of DHA-sulphate into Type I cyst fluids. Although the clinical significance remains to be determined, it demonstrates that breast epithelium may actively concentrate and retain substances from plasma. This means that breast epithelium may be exposed to high concentrations of xeno-biologicals for prolonged periods.
INTRODUCTION
concentrations are achieved, although a small study in which two patients were given radioactively labelled DHAsulphate did show accumulation of radioactivity in breast cyst fluids.’ The time course of this uptake and the differential uptake between the two types of cysts were not examined. The aim of the present study was to investigate more fully the uptake of DHA-sulphate into human breast cyst fluids.
Approximately 7% of all women in the Western world present to hospital with a clinically palpable breast cyst.’ The total number of cysts in individuals varies greatly. Approximately half of all patients develop a single cyst, one-third have from two to five cysts and the remainder develop more than five.’ Two populations of human breast cysts can be defined on the basis of their cyst fluid electrolyte composition and biochemistry. Type I cysts have a [sodium]/[potassium] ratio [Na+]/[K’]c3 and Type II cysts have a [Na’]/[K+]>3.2,3 The two populations are lined by different epithelium, Type I cysts being lined by apocrine and Type II cysts by flattened epithelium.4 These two cyst types also differ in the amounts of steroid hormones present within their cyst fluid.* For example, the mean concentration of the conjugated steroid, DHA-sulphate, found in Type I cysts is over loo-times greater than that in Type II cysts, which contain similar levels to those found in plasma? Little is known about the mechanism by which these Address Western
correspondence fo: J. M. Dixon, Edinburgh General Hospital, Crewe Road, Edinburgh
PATIENTS, MATERIALS
AND METHODS
Patients presenting to the Edinburgh Breast Unit with multiple palpable breast cysts were entered into the study. None were receiving medication for their condition and none were taking diuretics. Ethical permission for the investigation was obtained and the nature of the study explained to each patient before obtaining written consent. Over either 24 h (5 patients) or 48 h (11 patients), 1 MBq of 7-alpha tritiated (3H) DHA-sulphate was infused. Over the first hour, 10 ml of infusion was given and the remainder infused at a constant rate using an infusion pump. Blood and cyst fluids were obtained by venopuncture and needle
Breast Unit, EH4 2XU, UK
12
Uptake of dehydroepiandrosterone sulphate into human breast cyst fluids aspiration at varying time points during the period of infusion, while patients were sedated with intravenous Midazolam. In 15 patients the cyst fluids were aspirated during the period of infusion but in one patient who was infused over 48 h the cyst fluids were aspirated 7 days after the start of infusion. Blood was centrifuged at 2500 rpm for 10 min and the resultant plasma taken and frozen at -20°C. Cyst fluids were immediately stored at -20°C until batch analysis. The radioactivity in plasma and cyst fluids was determined by counting 25 ~1 and 250 ~1 aliquots in duplicate with and without the addition of 2000 dpm [3H] steroid (to monitor internal quenching) in 10 ml of NE260 scintillant. To confirm that radioactivity was associated with DHA-sulphate samples were extracted with ether and the aqueous phase subjected to acid solvolysis. The solvolysed material was extracted into ethyl acetate and run on thin layer chromatography together with authentic steroid standards. Electrolyte composition of cyst fluid was determined by flame photometry and cysts were classified into Type 1 if cyst fluid had a sodium:potassium ratio [Na’/K’] of <3 and were classified into Type II if the ratio was 23.‘S2,4 To compare uptake with sodium:potassium ratio, data from three patients infused over 48 h who had large numbers of cysts aspirated at 48-h time point were correlated. As the data varied over several orders of magnitude, the correlation was between the log of the ratio of sodium: potassium and the log of the radioactivity.
RESULTS Infusion over 24 h Representative results from two of the five patients infused are shown in Figure 1. Levels of radioactivity in plasma increased during the first few hours of infusion following the bolus but then remained constant so that between 20 and 24 h there was tight range of values for all five patients (3200-4700 dpm/ml). In contrast levels of radioactivity in cyst fluids tended to be low during the initial stages of infusion and in no patient was there more radioactivity in cysts fluids than in plasma during this early period. Cysts fluids aspirated at 20 h and beyond frequently contained high levels of radioactivity (Fig. 1). In certain patients levels of radioactivity were greater than those ever seen in plasma. A total of 60 cysts were aspirated from these five patients; 45 were Type I and 15 were Type II. Of these 66, 23 cysts contained greater levels of radioactivity than in plasma and all 23 were Type I cysts. There were 30 cysts aspirated at the 24-h time point and 17 of these contained higher levels of radioactivity than the corresponding level in plasma (Table 1). All fluids with excess radioactivity were Type I
13
Thousands 14 m
n
Type I I cysts
m
Type I cysts<
a dpmlml 6
2h
4h
A
6h
20h
24h
Time Thousands
15
dpmlml 10
/ B
-Type
Fig. 1 varying (B) Cyst infusion
II cysts
~Plasma
m
Type I cysts
(A) Levels of radioactivity in cyst fluids and plasma aspirated at times during a 24-h infusion (patient number 1 in Table 1). fluids and plasma aspirated at 24 h in a patient having a 24-h of radioactive DHA-sulphate (patient number 4 in Table 1).
cysts and none of the six Type II fluids aspirated at 24 h contained substantial amounts of radioactivity. Although four of the five women had cysts which contained more radioactivity than plasma, one patient (patient five) never accumulated more radioactivity in cyst fluid than in plasma.
Infusion over 48 h Representative results from two patients are shown in Figure 2. Levels of radioactivity in plasma were remarkably constant between 24 and 48 h and there was a narrow range between patients (1600-3500 dpm/ml). Levels of radio-
Table levels Patient
1 Proportion of cyst fluids containing in plasma as sampled at 24 h Type 1 fluids
Type II fluids
Excess
Excess
Total
Total
radioactivity
in excess Total Excess
Total
1 2 3 4 5
6 3 3 5 0
I 3 5 1 2
0 0 0 0 0
1 0 0 3 2
6 3 3 5 0
8 3 5 IO 4
Total
17
24
0
6
17
30
of
14
The Breast Table levels
Thousands 14 m
Plasma
Patient
6 6
0
24h
46h
Time
A
radioactivity
in excess
of
hsI Type I cysts
12 n
dpmlml
2 Proportion of cyst fluids containing in plasma as sampled at 48 h Type I fluids
Type II fluids
Total
Excess
Total
Excess
Total
Excess
Total
1 2 3 4 5 6 7 8 9 10
9 10 7 2 7 8 5 5 6 8
9 13 10 5 8 9 5 8 9 13
0 0 0 0 0 0 0 0 0 0
0 6 1 1 0 2 0 0 0 4
9 10 7 2 7 8 5 5 6 8
9 19 11 5 8 11 5 8 9 17
Total
67
89
0
14
67
103
14 12 10
dpm/ml
a 6 4 2 0
B
I
Type II cysts
~Plasma
6sI Type I cysts
Fig. 2 (A) Levels of radioactivity in cyst fluids and plasma at 24 and 48 h from a patient infused with radioactive DHA-sulphate over 48 h (patient number 1 in Table 2). (B) Levels of radioactivity in cyst fluids and plasma at 48 h in a patient having a 48-h infusion of radioactive DHA-sulphate (patient number 2 in Table 2).
activity in cyst fluids varied enormously from undetectable levels to in excess of 32 000 dpm/ml. The amount of radioactivity in cyst fluids tended to increase from 24 to 48 h. At 48 h the majority of cyst fluids contained more radioactivity than the corresponding level of plasma with some cyst fluids containing as much as 15fold greater excess than plasma. A total of 145 cysts were aspirated from the 10 patients whose cysts were aspirated during the 48-h period of infusion. Of these, 117 were Type I and 28 were Type II. Eighty-six cysts contained more radioactivity than the corresponding level in plasma and all 86 were Type I cysts. All patients by 48 h had at least some cysts which contained more radioactivity than the corresponding level in plasma (Table 2). Of a total of 103 cyst fluids aspirated at 48 h, 67 had levels of radioactivity higher than plasma (Table 2). All 67 were Type I and none of the 14 Type II cyst fluids accumulated radioactivity beyond that in plasma (Table 2).
Forty-eight-hour
infusion: cysts aspirated at 7 days
A total of five cysts were aspirated from this patient, three were Type I and two Type II (Table 3). The plasma level of
radioactivity at 7 days was low. Both Type II cysts aspirated had levels of radioactivity at 7 days which were in excess of those in plasma but lower than the circulating levels of radioactivity observed in other patients during the course of infusion. The three Type I cysts aspirated at 7 days contained much higher levels of radioactivity with a relative excess varying from 20 to 75-times the level in plasma at 7 days (Table 3). Cysts aspirated during follow-up During the 26-month period following the infusion with 7alpha tritiated DHA-sulphate a total of 39 cysts were aspirated from nine of the patients studied. The interval between infusion and aspiration varied between 3 weeks and 26 months. By 3 weeks the amount of radioactivity in plasma was negligible, but radioactive label could be detected in cyst fluids in samples taken over 2 years after infusion. Levels of radioactivity in Type I cyst fluids were much higher than those in Type II fluids. The levels of radioactivity in the 31 Type I cysts are plotted in Figure 3. Two patients were interesting in that the radioactivity found in cyst fluids aspirated 3 weeks and 3 months respectively after infusion were in excess of those in cyst fluids aspirated during the course of infusion.
Table 3 infusion
Radioactivity Breast
Plasma at 7 days cyst fluid
in cyst fluids
Type
and plasma
dpm
7 days after 48-h Relative
360 L L L R R
II II I I I
1440 960 26 960 8240 8120
4.0 2.6 74.9 22.9 22.6
excess
Uptake of dehydroepiandrosterone
sulphate into human breast cyst fluids
15
3 3 Ratio H in cyst fluid I H in plamla during infusion
. I.0
.
.
l
’
0.5
. l . . .
.‘, 0. 0.
. . . .
. . .
:: .*
Patient A3 .2 5
Fig. 3 Levels of radioactivity in breast cyst fluids aspirated intervals following infusion of radioactive DHA-sulphate.
Correlation between radioactivity with sodium:potassium ratio
01
at various Fig. 4 Correlation of log of radioactivity ratio. Data from three patients, analysing
with log of sodium:potassium only cysts aspirated at 48 h.
at 48-h compared
Data from three patients who had 43 cysts aspirated at 48 h after radioactive DHA-sulphate infusion showed that there was a direct correlation between the log of the sodium: potassium ratio and the log of the radioactivity (Fig. 4). This correlation was significant at FYO.001. In fact, there was a very clear inverse relationship and the majority of the points were close to a fitted 45” line with the exception of three cysts from a single patient.
Biochemical studies The tritium remained in the aqueous fraction after ether extraction (i.e. it was conjugated) and on acid solvolysis was extractable into an organic solvent. This extracted material had a similar chromatographic mobility to an authentic DHA standard on thin layer chromatography. This was true for cyst fluids taken during and after infusion and was irrespective of cyst type.
DISCUSSION The present observations demonstrate that 7-alpha tritiated DHA-sulphate appears in cyst fluids within hours of intravenous administration and that by 24 h the levels of radioactive DHA-sulphate in cyst fluids may exceed those in plasma. This active uptake appears to be specifically associated with Type I (apocrine) cysts and follow-up data suggest that the DHA-sulphate is retained within breast fluids over a prolonged period. The only other report of uptake of radioactive steroids into breast fluids was a limited study of two patients who had a bolus injection of labelled DHAsulphate following which one cyst was aspirated at three separate time points over a 48-h period.’ No details were subsequently given of the types of cysts aspirated or the
relative accumulation of radioactivity in cysts as compared to levels in plasma. By using patients with multiple cysts and aspirating fluids at different times during a constant intravenous infusion of radioactively labelled steroid it has been possible to study the extent and time course of the uptake of radioactively labelled DHA-sulphate into breast cysts. While the data demonstrate that there were differences between cysts in individual patients and that there was some variation from patient to patient, there was a general consistency of results which allows certain conclusions to be drawn. First, while levels of radioactively labelled DHAsulphate remained constant during the period of infusion, those in cyst fluids increased with time. Although radioactivity was detected in cyst fluids as early as 2 h, the levels were low and never exceeded those in plasma. In contrast, cysts aspirated at later time points frequently contained levels of radioactivity above those found in’ plasma and some cysts contained as much as 15times more radioactivity than in plasma. By the end of the infusion four of the five patients infused over 24 h and all the patients infused over 48 h had cysts which contained more radioactivity than plasma. The amounts of radioactive label in cyst fluids aspirated at 48 h were generally higher than those observed during the 24-h infusion. The further observation that the single patient who had a cyst aspiration delayed until one week after the completion of a 48-h infusion displayed among the highest levels of radioactivity in cyst fluids suggests that there is prolonged and persistent uptake of [3H] DHA-sulphate from the circulation. Although the majority of cysts accumulated [‘HI DHAsulphate in levels greater than that found in the circulation, a substantial number did not. When cysts were classified into Types I and II based on their electrolyte composition, only Type I cysts accumulated greater levels of radioactivity than plasma. The reason why some but not all Type I cysts accumulated radioactivity is uncertain but we were
16
The Breast
Table
4
Patient
Radioactivity Maximum levels plasma
in recurrent
cysts
Time of sample (h) cyst (h)
1
6880 (6)
11 960 (24)
4 5 2
4800 (6) 4760 (24) 2480 (48)
16 760 (24) 3800 (24) 20 280 (48)
6 7
2640 (48) 2960 (48)
12 120 (48) 11 240 (48)
8
3360 (48)
4280 (48)
10
3320 (48)
7400 (48)
11
2640 (48)
20 280 (48)
Recurrent sample
Type
Time of aspiration
590 530 510 1560 1840 1040 1720 400 185 120 5480 160 10040 1480 3160
I I I II I I I II II Plasma I Plasma I I I
18 months 18 months 18 months 3 months 3 months 3 months 3 months 3 weeks 3 weeks 3 weeks 3 weeks 3 weeks 3 months 6 months 18 months
unable to determine any other factor which was influential. The uptake of radioactive DHA-sulphate into Type I, but not Type II cysts, is compatible with our previous studies13294 which have demonstrated that endogenous levels of DHAsulphate in Type I cysts are on average loo-fold higher than those in the circulation, whereas Type II cysts show levels similar to those in plasma. It had previously been a matter of conjecture as to whether these high levels were due to in situ synthesis or derivation from plasma. The present study suggests that active uptake from plasma could account for these high levels. The clinical relevance of DHA-sulphate in cyst fluids has yet to be determined. For a hormone, DHA-sulphate is produced in large quantities6 and has relatively little biological activity unless metabolised.’ However, although an androgen in structure, at high concentrations it can behave as an oestrogen and induce oestrogenic responses.* DHA and its reduced metabolite, delta-5androstenediol can maintain the growth of oestrogen-dependent breast cancer cell lines.9~‘0It is thus of interest that the patients with Type I cysts have been shown to have an increased risk of breast cancer, as have women with apocrine metaplasia within their breast.” Furthermore, the accumulation of DHAsulphate may be a marker for other more active agents which could be accumulated by the same mechanism. For example, a recent report has described the accumulation of bile acids in breast cyst fluids.12 These observations indicate
the potential for breast epithelium to concentrate and retain xenobiologicals. Such prolonged exposure to noxious compounds could potentially explain the increased cancer risk which has been associated with Type I breast cystic disease.‘33’4
References 1. Dixon J M. Cystic disease of the breast. In: Smallwood, Taylor, eds. Benign breast disease. London: Edward Arnold, 1990; 66-84. 2. Miller W R, Dixon J M, Scott W N, Forrest A P M. Classification of human breast cysts according to electrolyte and androgen composition. Clin Oncol 1983; 9: 227-232. F D, Schwartz M K, Fleisher M. Cation 3. Bradlow H L, Skidmore levels in human breast cyst fluids. Clin Oncol 1981; 7: 388-390. 4. Dixon J M, Miller W R, Scott W N, Forrest A P M. The morphological basis of human breast populations. Br J Surg 1983; 70: 604-606. 5. Miller W R, Dixon J M, Forrest A P M. Hormonal correlates of apocrine secretion in the breast. Ann N Y Acad Sci 1986; 464: 275-287. 6. Abraham G E. Ovarian and adrenal contribution to peripheral antigens during the menstrual cycle. J Clin Endocrinol Met 1974; 39: 340-346. C, Dray F et al. An adrenal-secreted 7. Baulieu E E, Corpechot ‘androgen’; dehydroandrosterone sulphate, its metabolism and a tentative generalization of the metabolism of other steroid conjugates in man. Ret Prog Horm Res 1965; 21: 411-500. 8. Adams J, Garcia M, Rochefort H. Estrogenic effects of physiological concentrations of 5-androstene-3P, 17P-diol and its metabolism in MCF, human breast cancer cells. Cancer Res 1981; 41: 4720-4725. 9. Boccuzzi G, Brignardello E, Di Monaco M, Forte C, Leonardi I, Pizzini A. Influence of dehydroepiandrosterone and 5-en-androstene3-beta, 17 beta-diol on the growth of MCF-7 human breast-cancer cells induced by 17-beta-estradiol. Anticancer Res 1992; 12: 799-803. 10. Hackenberg R, Turgetto I, Filmer A, Schulz K-D. Estrogen and androgen receptor-mediated stimulation and inhibition of proliferation by androst-5-ene-3-beta, 17-beta-diol in human mammary cancer cells. J Steroid Biochem Molec Biol 1993; 46: 597-603. 11. Schuerch C, Rosen P P, Hirota T et al. A pathologic study of benign breast diseases in Tokyo and New York. Cancer 1982; 50: 1899-1903. 12. Javitt N B, Budai K, Miller D G, Cahan A C, Rasu N, Levitz M. Breast gut connection - origin of chenodeoxycholic acid in breast cyst fluid. Lancet 1994; 343: 633-635. 13. Miller W R, Scott W N, Harris W H, Wang D. Using biological measurements can patients with benign breast disease who are at high risk for breast cancer be identified. Cancer Det Prev 1992; 16: 99-106. 14. Angeli A, Dogliotti L, Naldoni C et al. Steroid biochemistry and categorization of breast cyst fluid - relation to breast cancer risk. J Steroid Biochem Mol Biol 1994; 49: 333-339.