This article was downloaded by: [New York University] On: 22 June 2015, At: 20:19 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Estrogenlc activities of chlorinated hydrocarbons J. Arly Nelson a
a b
a
, Robert F. Struck & Ruby James
a
Kettering‐Meyer Laboratory , Southern Research Institute , Birmingham, Alabama
b
Department of Pharmacology and Toxicology , University of Texas, Medical Branch , Galveston, Texas, 77550 Published online: 19 Oct 2009.
To cite this article: J. Arly Nelson , Robert F. Struck & Ruby James (1978) Estrogenlc activities of chlorinated hydrocarbons, Journal of Toxicology and Environmental Health: Current Issues, 4:2-3, 325-339, DOI: 10.1080/15287397809529664 To link to this article: http://dx.doi.org/10.1080/15287397809529664
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
ESTROGENlC ACTIVITIES OF CHLORINATED HYDROCARBONS J. Arly Nelson, Robert F. Struck, Ruby James
Downloaded by [New York University] at 20:19 22 June 2015
Kettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama
Some DDT analogs are estrogenic, particularly o,p'-DDT, which comprises approximately 15-20% of the commercial DDT mixture. Whether this compound or its metabolites are active has not been established. In fact, the data obtained thus far are more confusing than enlightening. For example, CC/4 pretreatment of immature female rats has been reported to inhibit or enhance estrogenic activity of o,p'-DDT, and SKF-525A pretreatment has been reported to enhance or not alter the estrogenic effect. Although o,p'-DDT inhibits binding of estradiol to the estrogen receptor from rat or human at low levels (~7-10 µM) in vitro, higher levels are required to inhibit nuclear binding of [ 3 H] estradiol in incubated whole uteri. Furthermore, o,p'-DDT appears to be neither estrogenic nor antiestrogenic in an in vitro estrogen assay. Methoxychlor appears to be "activated" by metabolism, and it is probable that phenolic metabolites are responsible for its estrogenic activity. Since chlorinated hydrocarbons often enhance the metabolism of steroids and may reduce circulating levels of steroids, interactions of the exogenous hormonaliy active agents with steroid receptors may be self-potentiating in vivo.
INTRODUCTION Since its introduction as an insecticide, DDT has had an impact on society that probably outweighs that of any other anthropogenic chemical (Metcalf, 1973). In spite of the development and utility of DDT, it is interesting to note that "more people die each year from mosquito-borne disease than from any other single cause" (Gillett, 1973). The commercial mixture of DDT contains about 15-20% o,p'-DDT, an isomer that has weak estrogenic properties (Balazs, 1976; Bitman et al., 1968; Bitman and Cecil, 1970; Clement and Okey, 1972; Gellert et al., 1972; Heinrichs et al., 1971; Singhal et al., 1970; Welch et al., 1969a). It has been estimated that as much as 200 million pounds of this substance has been released into the environment. Although the toxicity of DDT compounds to humans is not marked, it behooves a sophisticated society to learn as D. Garner and T. L. Barker provided expert technical assistance. Supported by project development funds of Southern Research Institute. J. A. Nelson's present address is Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77550. Requests for reprints should be sent to J. Arly Nelson, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77550.
325 Journal of Toxicology and Environmental Health, 4:325-339,1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/0402-032512.25
Downloaded by [New York University] at 20:19 22 June 2015
326
J. A. NELSON ETAL.
much as possible concerning the biochemical and pharmacological properties of agents so widely used and distributed. Apart from weak estrogenic effects of DDT analogs, p,p'-DDE has been implicated as a causal agent in eggshell thinning in certain sensitive birds (reviewed by Cooke, 1973), and DDT has been reported to be carcinogenic to mice (IARC, 1974). A complete understanding of the biochemical bases for these effects in animals other than humans should aid in the estimation of potential risks to society. For example, the implication that p,p'-DDE causes eggshell thinning through inhibition of Ca2+-ATPase (Miller et al., 1976) focuses attention on physiological systems in which this enzyme(s) plays important roles, whether one considers phytoplankton or humans. This review concerns the estrogenic activities of chlorinated hydrocarbons, particularly the relationships between structure, metabolism, and activity. 1 ' 2 An excellent, extensive review of pesticides and steroid interactions has been published by Kupfer (1975), and the estrogenic activities of pesticides other than DDT (e.g., Kepone, mirex, dieldrin, aldrin) have recently been further studied by Gellert (1977a, 1977b). Many other reviews of the metabolic and physiological effects of these agents related to hormone actions have appeared (e.g., Conney et al., 1967, 1973; Conney and Burns, 1972; Hrdina et al., 1975; Kupfer and Bulger, 1976a; Ware, 1975; Welch et al., 1969b). INTERACTIONS OF DDT ANALOGS WITH THE ESTROGEN RECEPTOR It is well established that an early event in the mechanism of estrogen response is the binding of the estrogenic substance with a cytoplasmic protein that is found in "target" tissues (reviewed by Jensen and DeSombre, 1972). DDT analogs evidently produce their estrogenic effect through interaction with the same receptor. The first suggestion that the DDT analogs bind to the receptor was that of Welch et al. (1969a), who observed that treatment of immature rats with o,p'-DDT and other active analogs in vivo reduced [ 3 H] estradiol uptake by the uterus, whereas less active analogs did not reduce the uptake. The interaction with the estrogen receptor was further confirmed by Cecil et al. (1971), who noted that either MER-25 or Actinomycin D (agents that interfere with estradiol 1 Preliminary reports of the original work reported here have appeared (Nelson et al., 1976, 1977). 2 Abbreviations used: p,p'-DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane; o,p'-DDT, 1,1,1-trichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl) ethane; p,p'-DDD, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane; m,p'-DDD, 1,1-dichloro-2-(p-chlorophenyl)-2-(m-chlorophenyl)ethane; o,p'-DDD, 1,1dichloro-2-(p-chIorophenyl)-2-(o-chlorophenyl)ethane; o,p'-DDE, 1,1-dichloro-2-(p-chlorophenyI)-2(o-chlorophenyl)ethene; p,p'-DDE, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethene; SKF-525A, 2-diethyIaminoethyl diphenylvalerate hydrochloride; and [ 3 H] estradiol, 17β-[6,7-3 H] estradiol.
Downloaded by [New York University] at 20:19 22 June 2015
CHLORINATED HYDROCARBONS
327
activity) prevented the estrogenlike effects of o,p'-DDT on the immature rat uterus. This observation is of special significance since MER-25 is a substance that acts as a competitor for estrogen-binding sites in the uterus. DDT analogs will compete for the binding of [3H]estradiol to the receptor present in rat uterine cytosol (Fig. 1). For a series of DDD analogs, the activity is o,p'- >m,p'- >p,p'-DDD. Side-chain modification in the o,p' isomer demonstrates apparent affinity for the receptor in the order D D T > D D D > D D E (Fig. 2). Inhibition of [3H]estradiol binding in vitro by these substances is correlated with uterotropic activity in vivo (Fig. 3). This finding suggests that metabolism is not required for the DDT analogs to interact with the receptor; however, whether the analogs have agonist activity per se remains to be established. The findings illustrated in Figs. 1 and 2 have been confirmed in at least three separate laboratories (Forster et al., 1974, 1975; Kupfer and Bulger, 1976a, 1976b; Nelson, 1974). Inhibition of [3H]estradiol binding to its receptor from tissues other than rat uterus (including human tissues) has also been observed (Kupfer and Bulger, 1977; Kupfer, 1975). Pretreatment of o,p'-DDT with liver microsomes under conditions that should allow mixed-function oxidase activity (Hill et al., 1972) did not enhance the apparent affinity of o,p'-DDT for the estrogen receptor (Table 1). This was also true for p,p'-DDT and two polychlorinated biphenyl mixtures, Aroclor 1221 and Aroclor 1254. On the other hand, methoxychlor and technical methoxychlor (one experiment) were more effective as 80r-
i,p- DDD
•I
I" 1 " K) 100 Concentration, fiM
FIGURE 1. Inhibition of [ 3 H]estradiol binding to rat uterine receptor protein by DDD isomers. Mean values ± SE are shown (adapted from Nelson, 1974).
IOO r
O,p'-DDT
Downloaded by [New York University] at 20:19 22 June 2015
,p'-DDT
P.P'-DDD P, p1- DDE .01 Concentration, FIGURE 2. Inhibition by DDT analogs of [ 3 H] estradiol binding to rat uterine receptor protein. The values shown are mean + SE for at least three determinations (adapted from Nelson, 1974).
10
20
30
40
50
100
•100
!80-
#o,p'-DOT
OQ
60
60
-80
-60
' •
• o,p'-DO£
"5 40H
-40 m,p'-DDD %f
20-
-20 pp'-DCO and p,p'-DDE (S)
10
20
30
40
50
60
% Increase Uterine Weight
FIGURE 3. Correlation between inhibition of [ 3 H]estradiol binding in vitro and uterotropic effect in vivo for the agents shown in Figs. 1 and 2. Inhibition of in vitro [ 3 H]estradiol binding, as measured at the highest concentrations shown in Figs. 1 and 2, is plotted against the increase in uterine wet weight of immature rats treated 6 h earlier with doses of 50 mg/kg jp (Welch et al., 1969a). [ 3 H] Estradiol binding in vitro to rat uterine cytosoi protein was determined by using the dextran-coated charcoal procedure described previously (Nelson, 1974).
328
CHLORINATED HYDROCARBONS
329
TABLE 1. Effect of Activation by Liver Microsomes on the Inhibition by Chlorinated Hydrocarbons of [ 3 H] Estradiol Binding to Rat Uterus Receptor0
Downloaded by [New York University] at 20:19 22 June 2015
% Inhibition of binding Agent
+TPNH
-TPNH
A± SE
Microsomes only Microsomes plus Methoxychlor Technical methoxychlor o,p'-DDT p,p'-DDT Aroclor 1221 Aroclor 1254
2
-3
-1±26
51 48 22 0 11 0
4 0 17 3 4 8
47 ± 1° 4 8 d
5±Ab
H
d
-3
7±Sb
~&d
°The agents shown were incubated with rat liver microsomes (25 mg equivalent wet weight of liver) for 1 h in the presence or absence of 2.3 m/W TPNH at 37°C (Hill et al., 1972). After stopping the reaction by heating at 100°C for 2 min, 50 MI were tested for inhibition of [ 3 H]estradiol binding as previously described (Nelson, 1974). Calculated concentrations of the agents and ethanol were: microsomal incubation, 50 ppm and 4%; binding assay, 10 ppm and 0.8%. "Not significantly different from o,p > 0.05, n = 4. Significantly different from o,p < 0.01, n = 4.
inhibitors of [ 3 H] estradiol binding following the liver microsomal pretreatment (Table 1). This suggests that metabolites of methoxychlor may be formed that have a higher affinity for the receptor than does methoxychlor. In one such incubation, a metabolite (structure I in Fig. 4) was verified by subsequent gas chromatography and mass spectrometry of the reaction mixture (Fig. 5). Methoxychlor has been reported to be progressively demethylated to the mono- and bis-phenol derivatives (structures I and II in Fig. 4) in vitro and in vivo (Kapoor et al., 1970). Metabolism to phenolic products that are structurally related to diethylstilbestrol suggests that metabolism of methoxychlor is required for estrogenic activity. Methoxychlor is relatively inactive as an inhibitor of [3H]estradiol binding in vitro (Table 1; Nelson, 1974), and a recent report suggests that the low activity resides in phenolic impurities that can be removed with base treatment (Bulger and Kupfer, 1977). In summary, metabolism of o,p'-DDT and its active analogs (with the possible exception of methoxychlor) is not required for inhibition of [ 3 H] estradiol receptor-specific binding in vitro. Also, the apparent affinities of DDT analogs for the estrogen receptor are correlated with their in vivo activities (Fig. 3).
330
J. A. NELSON E T A L .
METHOXYCHLOR
DIETHYLSTILBESTROL
Downloaded by [New York University] at 20:19 22 June 2015
FIGURE 4. Structures of methoxychlor, two of its major metabolites, and diethylstilbestrol.
METABOLISM OF DDT ANALOGS RELATED TO THEIR ESTROGENIC ACTIVITIES The estrogenlike activity of DDT analogs has often been ascribed to their structural relationship to diethylstilbestrol (e.g., Bitman and Cecil, 1970; Fisher et al., 1952). In the diethylstilbestrol series, a phenolic
RETENTION T I M E , MIN.
FIGURE 5. Gas chromatographic (GC) analysis of methoxychlor after incubation w i t h rat liver microsomes. Reaction mixtures of samples given in Table 1 were extracted with cyclohexane f o r GC analysis. The metabolite was identified (structure I, Fig. 4) by mass spectra obtained with the GC effluent, using a Hitachi high-resolution, double-focusing RMU-6-D-3 mass spectrometer. Column, 4 f t X 0.25 in O D glass; packing, 3.8% UX-W98 on 80/100 Chromosorb W; column temperature, 210°C; injection port temperature, 240°C; detector, electron capture, 270°C; carrier gas, helium, 50 m l / m i n .
Downloaded by [New York University] at 20:19 22 June 2015
CHLORINATED HYDROCARBONS
331
hydroxyl group has generally been observed to be necessary for hormonal activity (Solmssen, 1945). It was not surprising, therefore, that pretreatment of immature rats with CCI4 reduced the uterotropic activity of o,p'-DDT (Welch et al., 1969a), since this pretreatment should reduce the rate of metabolism of o,p'-DDT. Welch et al. (1969a) also reported that SKF-525A pretreatment did not alter the uterotropic effect of o,p'-DDT. Since metabolism did not appear necessary for interaction of o,p'-DDT with the estrogen receptor (Fig. 2), we have reexamined the effects of CCI4 in vivo on the uterotropic effect of o,p'-DDT. In addition, another inhibitor of liver mixed-function oxidase activity, SKF-525A, and an inducer, phenobarbital, were employed. Pretreatment with CCI4 or SKF-525A appeared to enhance the o,p'-DDT effect, whereas phenobarbital pretreatment reduced the effect (Fig. 6). In separate but simultaneous experiments, these pretreatments altered the sleeping times of the rats, as expected from their well-established effects on drug-metabolizing enzymes (legend to Fig. 6). Pretreatment with CCI4 enhanced the uterotropic effect of methoxychlor; however, SKF-525A or phenobarbital pretreatment did not alter the methoxychlor response (Fig. 6). The observation that CCI4 pretreatment enhanced rather than reduced the response to o,p'-DDT is in direct contrast to that reported by Welch et O,P'-DDT. 10 MG/KG
MCTHOXYCHLOR, 50 MG/KG
1.50-
(13)
(21) (15)
(16)
(II) (II)
1.40CCI,
(9)
CCI4
1.30-
1.20-
1.10-
P H N 0 B A R
NO "DRUG
1.00-
FIGURE 6. Effect of inhibition or induction of liver microsomal enzymes on the uterotropic response to o,p'-DDT or to methoxychlor. Immature rats were pretreated with CCI4 (0.67 ml/kg, 24 h), SKF-525A (25 mg/kg, 30 min), phenobarbital (50 mg/kg, once daily for 3 d), or appropriate vehicle (control). At 6-8 h after administration of o,p'-DDT or methoxychlor, uteri were removed and weighed. The results shown are mean values ± SE. Pentobarbital (32 mg/kg) sleeping times (in minutes, mean ± SE) of rats thus treated were: control group, 86 ± 4; CCI4 group, 150 ± 4 ; SKF-525A group, 135 ± 7; phenobarbital group,
Journal of Toxicology and Environmental Health: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh19
Estrogenlc activities of chlorinated hydrocarbons J. Arly Nelson a
a b
a
, Robert F. Struck & Ruby James
a
Kettering‐Meyer Laboratory , Southern Research Institute , Birmingham, Alabama
b
Department of Pharmacology and Toxicology , University of Texas, Medical Branch , Galveston, Texas, 77550 Published online: 19 Oct 2009.
To cite this article: J. Arly Nelson , Robert F. Struck & Ruby James (1978) Estrogenlc activities of chlorinated hydrocarbons, Journal of Toxicology and Environmental Health: Current Issues, 4:2-3, 325-339, DOI: 10.1080/15287397809529664 To link to this article: http://dx.doi.org/10.1080/15287397809529664
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
ESTROGENlC ACTIVITIES OF CHLORINATED HYDROCARBONS J. Arly Nelson, Robert F. Struck, Ruby James
Downloaded by [New York University] at 20:19 22 June 2015
Kettering-Meyer Laboratory, Southern Research Institute, Birmingham, Alabama
Some DDT analogs are estrogenic, particularly o,p'-DDT, which comprises approximately 15-20% of the commercial DDT mixture. Whether this compound or its metabolites are active has not been established. In fact, the data obtained thus far are more confusing than enlightening. For example, CC/4 pretreatment of immature female rats has been reported to inhibit or enhance estrogenic activity of o,p'-DDT, and SKF-525A pretreatment has been reported to enhance or not alter the estrogenic effect. Although o,p'-DDT inhibits binding of estradiol to the estrogen receptor from rat or human at low levels (~7-10 µM) in vitro, higher levels are required to inhibit nuclear binding of [ 3 H] estradiol in incubated whole uteri. Furthermore, o,p'-DDT appears to be neither estrogenic nor antiestrogenic in an in vitro estrogen assay. Methoxychlor appears to be "activated" by metabolism, and it is probable that phenolic metabolites are responsible for its estrogenic activity. Since chlorinated hydrocarbons often enhance the metabolism of steroids and may reduce circulating levels of steroids, interactions of the exogenous hormonaliy active agents with steroid receptors may be self-potentiating in vivo.
INTRODUCTION Since its introduction as an insecticide, DDT has had an impact on society that probably outweighs that of any other anthropogenic chemical (Metcalf, 1973). In spite of the development and utility of DDT, it is interesting to note that "more people die each year from mosquito-borne disease than from any other single cause" (Gillett, 1973). The commercial mixture of DDT contains about 15-20% o,p'-DDT, an isomer that has weak estrogenic properties (Balazs, 1976; Bitman et al., 1968; Bitman and Cecil, 1970; Clement and Okey, 1972; Gellert et al., 1972; Heinrichs et al., 1971; Singhal et al., 1970; Welch et al., 1969a). It has been estimated that as much as 200 million pounds of this substance has been released into the environment. Although the toxicity of DDT compounds to humans is not marked, it behooves a sophisticated society to learn as D. Garner and T. L. Barker provided expert technical assistance. Supported by project development funds of Southern Research Institute. J. A. Nelson's present address is Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77550. Requests for reprints should be sent to J. Arly Nelson, Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas 77550.
325 Journal of Toxicology and Environmental Health, 4:325-339,1978 Copyright © 1978 by Hemisphere Publishing Corporation 0098-4108/78/0402-032512.25
Downloaded by [New York University] at 20:19 22 June 2015
326
J. A. NELSON ETAL.
much as possible concerning the biochemical and pharmacological properties of agents so widely used and distributed. Apart from weak estrogenic effects of DDT analogs, p,p'-DDE has been implicated as a causal agent in eggshell thinning in certain sensitive birds (reviewed by Cooke, 1973), and DDT has been reported to be carcinogenic to mice (IARC, 1974). A complete understanding of the biochemical bases for these effects in animals other than humans should aid in the estimation of potential risks to society. For example, the implication that p,p'-DDE causes eggshell thinning through inhibition of Ca2+-ATPase (Miller et al., 1976) focuses attention on physiological systems in which this enzyme(s) plays important roles, whether one considers phytoplankton or humans. This review concerns the estrogenic activities of chlorinated hydrocarbons, particularly the relationships between structure, metabolism, and activity. 1 ' 2 An excellent, extensive review of pesticides and steroid interactions has been published by Kupfer (1975), and the estrogenic activities of pesticides other than DDT (e.g., Kepone, mirex, dieldrin, aldrin) have recently been further studied by Gellert (1977a, 1977b). Many other reviews of the metabolic and physiological effects of these agents related to hormone actions have appeared (e.g., Conney et al., 1967, 1973; Conney and Burns, 1972; Hrdina et al., 1975; Kupfer and Bulger, 1976a; Ware, 1975; Welch et al., 1969b). INTERACTIONS OF DDT ANALOGS WITH THE ESTROGEN RECEPTOR It is well established that an early event in the mechanism of estrogen response is the binding of the estrogenic substance with a cytoplasmic protein that is found in "target" tissues (reviewed by Jensen and DeSombre, 1972). DDT analogs evidently produce their estrogenic effect through interaction with the same receptor. The first suggestion that the DDT analogs bind to the receptor was that of Welch et al. (1969a), who observed that treatment of immature rats with o,p'-DDT and other active analogs in vivo reduced [ 3 H] estradiol uptake by the uterus, whereas less active analogs did not reduce the uptake. The interaction with the estrogen receptor was further confirmed by Cecil et al. (1971), who noted that either MER-25 or Actinomycin D (agents that interfere with estradiol 1 Preliminary reports of the original work reported here have appeared (Nelson et al., 1976, 1977). 2 Abbreviations used: p,p'-DDT, 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane; o,p'-DDT, 1,1,1-trichloro-2-(p-chlorophenyl)-2-(o-chlorophenyl) ethane; p,p'-DDD, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane; m,p'-DDD, 1,1-dichloro-2-(p-chlorophenyl)-2-(m-chlorophenyl)ethane; o,p'-DDD, 1,1dichloro-2-(p-chIorophenyl)-2-(o-chlorophenyl)ethane; o,p'-DDE, 1,1-dichloro-2-(p-chlorophenyI)-2(o-chlorophenyl)ethene; p,p'-DDE, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethene; SKF-525A, 2-diethyIaminoethyl diphenylvalerate hydrochloride; and [ 3 H] estradiol, 17β-[6,7-3 H] estradiol.
Downloaded by [New York University] at 20:19 22 June 2015
CHLORINATED HYDROCARBONS
327
activity) prevented the estrogenlike effects of o,p'-DDT on the immature rat uterus. This observation is of special significance since MER-25 is a substance that acts as a competitor for estrogen-binding sites in the uterus. DDT analogs will compete for the binding of [3H]estradiol to the receptor present in rat uterine cytosol (Fig. 1). For a series of DDD analogs, the activity is o,p'- >m,p'- >p,p'-DDD. Side-chain modification in the o,p' isomer demonstrates apparent affinity for the receptor in the order D D T > D D D > D D E (Fig. 2). Inhibition of [3H]estradiol binding in vitro by these substances is correlated with uterotropic activity in vivo (Fig. 3). This finding suggests that metabolism is not required for the DDT analogs to interact with the receptor; however, whether the analogs have agonist activity per se remains to be established. The findings illustrated in Figs. 1 and 2 have been confirmed in at least three separate laboratories (Forster et al., 1974, 1975; Kupfer and Bulger, 1976a, 1976b; Nelson, 1974). Inhibition of [3H]estradiol binding to its receptor from tissues other than rat uterus (including human tissues) has also been observed (Kupfer and Bulger, 1977; Kupfer, 1975). Pretreatment of o,p'-DDT with liver microsomes under conditions that should allow mixed-function oxidase activity (Hill et al., 1972) did not enhance the apparent affinity of o,p'-DDT for the estrogen receptor (Table 1). This was also true for p,p'-DDT and two polychlorinated biphenyl mixtures, Aroclor 1221 and Aroclor 1254. On the other hand, methoxychlor and technical methoxychlor (one experiment) were more effective as 80r-
i,p- DDD
•I
I" 1 " K) 100 Concentration, fiM
FIGURE 1. Inhibition of [ 3 H]estradiol binding to rat uterine receptor protein by DDD isomers. Mean values ± SE are shown (adapted from Nelson, 1974).
IOO r
O,p'-DDT
Downloaded by [New York University] at 20:19 22 June 2015
,p'-DDT
P.P'-DDD P, p1- DDE .01 Concentration, FIGURE 2. Inhibition by DDT analogs of [ 3 H] estradiol binding to rat uterine receptor protein. The values shown are mean + SE for at least three determinations (adapted from Nelson, 1974).
10
20
30
40
50
100
•100
!80-
#o,p'-DOT
OQ
60
60
-80
-60
' •
• o,p'-DO£
"5 40H
-40 m,p'-DDD %f
20-
-20 pp'-DCO and p,p'-DDE (S)
10
20
30
40
50
60
% Increase Uterine Weight
FIGURE 3. Correlation between inhibition of [ 3 H]estradiol binding in vitro and uterotropic effect in vivo for the agents shown in Figs. 1 and 2. Inhibition of in vitro [ 3 H]estradiol binding, as measured at the highest concentrations shown in Figs. 1 and 2, is plotted against the increase in uterine wet weight of immature rats treated 6 h earlier with doses of 50 mg/kg jp (Welch et al., 1969a). [ 3 H] Estradiol binding in vitro to rat uterine cytosoi protein was determined by using the dextran-coated charcoal procedure described previously (Nelson, 1974).
328
CHLORINATED HYDROCARBONS
329
TABLE 1. Effect of Activation by Liver Microsomes on the Inhibition by Chlorinated Hydrocarbons of [ 3 H] Estradiol Binding to Rat Uterus Receptor0
Downloaded by [New York University] at 20:19 22 June 2015
% Inhibition of binding Agent
+TPNH
-TPNH
A± SE
Microsomes only Microsomes plus Methoxychlor Technical methoxychlor o,p'-DDT p,p'-DDT Aroclor 1221 Aroclor 1254
2
-3
-1±26
51 48 22 0 11 0
4 0 17 3 4 8
47 ± 1° 4 8 d
5±Ab
H
d
-3
7±Sb
~&d
°The agents shown were incubated with rat liver microsomes (25 mg equivalent wet weight of liver) for 1 h in the presence or absence of 2.3 m/W TPNH at 37°C (Hill et al., 1972). After stopping the reaction by heating at 100°C for 2 min, 50 MI were tested for inhibition of [ 3 H]estradiol binding as previously described (Nelson, 1974). Calculated concentrations of the agents and ethanol were: microsomal incubation, 50 ppm and 4%; binding assay, 10 ppm and 0.8%. "Not significantly different from o,p > 0.05, n = 4. Significantly different from o,p < 0.01, n = 4.
inhibitors of [ 3 H] estradiol binding following the liver microsomal pretreatment (Table 1). This suggests that metabolites of methoxychlor may be formed that have a higher affinity for the receptor than does methoxychlor. In one such incubation, a metabolite (structure I in Fig. 4) was verified by subsequent gas chromatography and mass spectrometry of the reaction mixture (Fig. 5). Methoxychlor has been reported to be progressively demethylated to the mono- and bis-phenol derivatives (structures I and II in Fig. 4) in vitro and in vivo (Kapoor et al., 1970). Metabolism to phenolic products that are structurally related to diethylstilbestrol suggests that metabolism of methoxychlor is required for estrogenic activity. Methoxychlor is relatively inactive as an inhibitor of [3H]estradiol binding in vitro (Table 1; Nelson, 1974), and a recent report suggests that the low activity resides in phenolic impurities that can be removed with base treatment (Bulger and Kupfer, 1977). In summary, metabolism of o,p'-DDT and its active analogs (with the possible exception of methoxychlor) is not required for inhibition of [ 3 H] estradiol receptor-specific binding in vitro. Also, the apparent affinities of DDT analogs for the estrogen receptor are correlated with their in vivo activities (Fig. 3).
330
J. A. NELSON E T A L .
METHOXYCHLOR
DIETHYLSTILBESTROL
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FIGURE 4. Structures of methoxychlor, two of its major metabolites, and diethylstilbestrol.
METABOLISM OF DDT ANALOGS RELATED TO THEIR ESTROGENIC ACTIVITIES The estrogenlike activity of DDT analogs has often been ascribed to their structural relationship to diethylstilbestrol (e.g., Bitman and Cecil, 1970; Fisher et al., 1952). In the diethylstilbestrol series, a phenolic
RETENTION T I M E , MIN.
FIGURE 5. Gas chromatographic (GC) analysis of methoxychlor after incubation w i t h rat liver microsomes. Reaction mixtures of samples given in Table 1 were extracted with cyclohexane f o r GC analysis. The metabolite was identified (structure I, Fig. 4) by mass spectra obtained with the GC effluent, using a Hitachi high-resolution, double-focusing RMU-6-D-3 mass spectrometer. Column, 4 f t X 0.25 in O D glass; packing, 3.8% UX-W98 on 80/100 Chromosorb W; column temperature, 210°C; injection port temperature, 240°C; detector, electron capture, 270°C; carrier gas, helium, 50 m l / m i n .
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CHLORINATED HYDROCARBONS
331
hydroxyl group has generally been observed to be necessary for hormonal activity (Solmssen, 1945). It was not surprising, therefore, that pretreatment of immature rats with CCI4 reduced the uterotropic activity of o,p'-DDT (Welch et al., 1969a), since this pretreatment should reduce the rate of metabolism of o,p'-DDT. Welch et al. (1969a) also reported that SKF-525A pretreatment did not alter the uterotropic effect of o,p'-DDT. Since metabolism did not appear necessary for interaction of o,p'-DDT with the estrogen receptor (Fig. 2), we have reexamined the effects of CCI4 in vivo on the uterotropic effect of o,p'-DDT. In addition, another inhibitor of liver mixed-function oxidase activity, SKF-525A, and an inducer, phenobarbital, were employed. Pretreatment with CCI4 or SKF-525A appeared to enhance the o,p'-DDT effect, whereas phenobarbital pretreatment reduced the effect (Fig. 6). In separate but simultaneous experiments, these pretreatments altered the sleeping times of the rats, as expected from their well-established effects on drug-metabolizing enzymes (legend to Fig. 6). Pretreatment with CCI4 enhanced the uterotropic effect of methoxychlor; however, SKF-525A or phenobarbital pretreatment did not alter the methoxychlor response (Fig. 6). The observation that CCI4 pretreatment enhanced rather than reduced the response to o,p'-DDT is in direct contrast to that reported by Welch et O,P'-DDT. 10 MG/KG
MCTHOXYCHLOR, 50 MG/KG
1.50-
(13)
(21) (15)
(16)
(II) (II)
1.40CCI,
(9)
CCI4
1.30-
1.20-
1.10-
P H N 0 B A R
NO "DRUG
1.00-
FIGURE 6. Effect of inhibition or induction of liver microsomal enzymes on the uterotropic response to o,p'-DDT or to methoxychlor. Immature rats were pretreated with CCI4 (0.67 ml/kg, 24 h), SKF-525A (25 mg/kg, 30 min), phenobarbital (50 mg/kg, once daily for 3 d), or appropriate vehicle (control). At 6-8 h after administration of o,p'-DDT or methoxychlor, uteri were removed and weighed. The results shown are mean values ± SE. Pentobarbital (32 mg/kg) sleeping times (in minutes, mean ± SE) of rats thus treated were: control group, 86 ± 4; CCI4 group, 150 ± 4 ; SKF-525A group, 135 ± 7; phenobarbital group,
