Equivalence of cell survival data for radiation dose and thermal dose in ablative treatments: analysis applied to essential tremor thalamotomy by focused ultrasound and gamma knife.

Thermal dose and absorbed radiation dose have historically been difficult to compare because different biological mechanisms are at work. Thermal dose denatures proteins and the radiation dose causes DNA damage in order to achieve ablation. The purpose of this paper is to use the proportion of cell survival as a potential common unit by which to measure the biological effect of each procedure. Survival curves for both thermal and radiation doses have been extracted from previously published data for three different cell types. Fits of these curves were used to convert both thermal and radiation dose into the same quantified biological effect: fraction of surviving cells. They have also been used to generate and compare survival profiles from the only indication for which clinical data are available for both focused ultrasound (FUS) thermal ablation and radiation ablation: essential tremor thalamotomy. All cell types could be fitted with coefficients of determination greater than 0.992. As an illustration, survival profiles of clinical thalamotomies performed by radiosurgery and FUS are plotted on a same graph for the same metric: fraction of surviving cells. FUS and Gamma Knife have the potential to be used in combination to deliver a more effective treatment (for example, FUS may be used to debulk the main tumour mass, and radiation to treat the surrounding tumour bed). In this case, a model which compares thermal and radiation treatments is valuable in order to adjust the dose between the two.

Radiation effects on DNA synthesis in a defined chromosomal replicon.

It has recently been shown that the tumor suppressor p53 mediates a signal transduction pathway that responds to DNA damage by arresting cells in the late G1 period of the cell cycle. However, the operation of this pathway alone cannot explain the 50% reduction in the rate of DNA synthesis that occurs within 30 min of irradiation of an asynchronous cell population. We are using the amplified dihydrofolate reductase (DHFR) domain in the methotrexate-resistant CHO cell line, CHOC 400, as a model replicon in which to study this acute radiation effect. We first show that the CHOC 400 cell line retains the classical acute-phase response but does not display the late G1 arrest that characterizes the p53-mediated checkpoint. Using a two-dimensional gel replicon-mapping method, we then show that when asynchronous cultures are irradiated with 900 cGy, initiation in the DHFR locus is completely inhibited within 30 min and does not resume for 3 to 4 h. Since initiation in this locus occurs throughout the first 2 h of the S period, this result implies the existence of a p53-independent S-phase damage-sensing pathway that functions at the level of individual origins. Results obtained with the replication inhibitor mimosine define a position near the G1/S boundary beyond which cells are unable to prevent initiation at early-firing origins in response to irradiation. This is the first direct demonstration at a defined chromosomal origin that radiation quantitatively down-regulates initiation.

Histone H1 and H3 dephosphorylation are differentially regulated by radiation-induced signal transduction pathways.

We recently demonstrated that linker histone H1, which is thought to have a fundamental role in higher-order chromatin structure, becomes transiently dephosphorylated after ionizing radiation (IR) in a mutated ataxia telangiectasia (ATM) dependent manner. To establish whether H1 dephosphorylation was a component of a damage-response pathway that included dephosphorylation of other histones, we asked whether H3 was dephosphorylated in response to IR in a manner similar to H1. H1 and H3 are maximally phosphorylated in metaphase and both are dephosphorylated after IR. However, the duration of IR-induced H3 dephosphorylation is significantly longer than that of IR-induced H1 dephosphorylation. Moreover, H1 dephosphorylation is ATM-dependent, whereas H3 dephosphorylation is ATM-independent. These observations suggest that the damage-sensing pathways regulating H3 and H1 dephosphorylation diverge upstream of ATM.

The clearance and metabolism of estradiol and estradiol-17-esters in the rat.

The C-17 fatty acid esters of estradiol are naturally occurring estrogens which have been shown to circulate in blood. They are long-acting estrogens, analogous to the synthetic alkyl and aryl esters of estrogens which have been used pharmacologically for decades. To determine the mechanisms involved in the prolonged stimulation evoked by these nonpolar estrogens, several C-17 alkyl esters were synthesized and labeled with 3H at C-17 alpha, and their metabolism and clearance were studied and compared to those of estradiol in rats. The conversion of the C-17-3H to 3H2O was used as a marker of metabolism. While the clearance of the long chain esters from blood is somewhat slower than that of estradiol (t 1/2 = approximately 16 vs. 2 min, respectively), the rates of metabolism are dramatically different. For example, the t 1/2 of metabolism for two representative esters, estradiol-17-stearate and arachidonate, are 580 and 365 min, respectively, while the t 1/2 of metabolism for estradiol is about as fast as its clearance from blood (approximately 2 min). When the effect of chain length was studied, it was found that for the smaller esters, there was an inverse relationship between the size of the acyl group and the clearance from blood, i.e. the longer carboxylic acids were cleared more slowly. However, when the acyl group was lengthened from C12 to C14, the rate of clearance increased and was even faster with C18. Nevertheless, with all of the esters tested, the rate of metabolism steadily decreased as the chain length increased. These results are interpreted as indicating that the control point or rate-limiting step in the metabolism of the estradiol esters is the esterase that hydrolyzes the ester to estradiol. Thus, the prolonged estrogenic action of the C-17-alkyl esters is due to the slow release of estradiol from this hydrophobic reservoir.

Binding of estradiol-17-fatty acid esters to plasma proteins.

The C-17 fatty acid esters of estradiol are a unique family of long-acting estrogens that circulate in blood. The unusual duration of the estrogenic action of these esters has been shown previously to correlate with their very slow rate of metabolism. However, in striking contrast to their slow rate of metabolism, the clearance of these esters from blood is relatively rapid, not very different from that of estradiol (E2). Studies on the effect of the size of the carboxylic acid moiety on the rates of both metabolism and clearance have suggested that an active process might be involved in the cellular uptake of these circulating esters, and this, in turn, raised the question of how E2-fatty acid esters are transported in blood. The binding of representative E2-17-fatty acid esters to both human and rat plasma proteins known to bind either E2 or fatty acids was investigated. As expected, both E2 and 5 alpha-dihydrotestosterone bound to human sex hormone-binding globulin, whereas none of the E2 esters bound to this human plasma protein. Similarly, E2 bound to rat alpha-fetoprotein (AFP) and unsaturated fatty acids bound to both human and rat AFP, but none of the E2 esters bound to AFP of either species. These steroid esters bind to lipoproteins. Over 85% of a representative ester, E2-17-stearate, partitioned in the lipoprotein fractions of both human and rat serum, while the synthetic short chain ester, E2-17 beta-acetate, like E2 itself, partitioned predominantly in the nonlipoprotein fraction of blood. These results demonstrate the unusual binding of a family of steroid hormones to plasma lipoproteins and open the possibility that they are transported into target cells through the mediation of lipoprotein receptors.

The interaction of C-17 esters of estradiol with the estrogen receptor.

Various C-17 alkyl esters of estradiol (E2) were tested as ligands for the estrogen receptor. The naturally occurring fatty acid esters, represented by E2-17-stearate, E2-17-palmitate, and E2-17-arachidonate, did not compete with [3H]E2 for receptor-binding sites. [3H]E2-17-stearate did not bind in a specific or saturable manner to uterine cytosol. On the other hand, the long-acting pharmacological esters of E2, E2-17-acetate, -propionate, -valerate, etc., were highly effective in competing for the binding of [3H]E2 to the uterine receptor. However, it was found that these short chain esters were hydrolyzed to E2 when incubated with uterine cytosol under the conditions used for the binding assay. Consequently, [3H]E2-17-acetate and [3H] E2-17-valerate were tested as ligands in an assay in which the hydrolytic activity was minimized by partially purifying the receptor with (NH4)2SO4 precipitation and by limiting the duration of the incubation. In this assay there was no specific binding of the C-17-valerate ester. There was a relatively small amount of specifically bound radioactivity in the incubation of the acetate ester, but upon high performance liquid chromatographic analysis, the specifically bound steroid was found to be E2 and not the ester. These results indicate that the C-17 esters of E2, from C2 to the long chain fatty acids, all of which are potent long-acting estrogens, exert their estrogenic effects only after hydrolysis to the free C18 steroid.

Actions of an estradiol-17-fatty acid ester in estrogen target tissues of the rat: comparison with other C-17 metabolites and a pharmacological C-17 ester.

The C-17 fatty acid esters of estradiol (E2) are a unique family of nonpolar estradiol metabolites. They are potent long-acting estrogens that represent the natural analog of the synthetic esters used for estrogen therapy. We measured the uterotropic response and the formation of uterine nuclear estrogen receptors (ERn) produced by iv administration of a representative ester, E2-17-stearate, in comparison to E2, other natural C-17 conjugates of E2, E2-17-glucuronide, and E2-17-sulfate, and the pharmacological ester E2-17-cyclopentylpropionate. While E2-17-stearate produced a sustained and greater uterotropic response compared to E2, the maximal induction of ERn by the ester was only about one third of that induced by a similar dose of E2. However, the induction of ERn by E2-17-stearate was markedly sustained compared to that by E2. Furthermore, the initiation of the ERn response to E2-17-stearate was delayed. Since E2-17-esters do not bind to the ER, this delay is consistent with the requirement for hydrolysis of the esters before interaction with the ER. Neither of the ionic conjugates of E2 (sulfate and glucuronide) produced an increase in ERn concentrations or a uterotropic response. The synthetic ester cyclopentylpropionate, like E2, produced a rapid ERn response and a significantly shorter uterotropic response than the stearate ester. When the induction of ERn by E2-17-stearate was investigated in other target tissues there were no marked differences in the brain, pituitary, and liver. No blood-brain barrier was apparent for the formation of ERn, despite the fact that this steroidal ester circulates in the blood bound to lipoproteins. These findings suggest that this unusual family of steroidal esters has biological properties that differentiate them from other known estrogens, natural and synthetic, in terms of their ability to produce a slow-onset sustained estrogenic stimulus in a variety of different estrogen target tissues.

Biological activity of the fatty acid ester metabolites of corticoids.

Fatty acid esters of all families of steroid hormones are known to exist naturally. While their physiological roles are not clear, the C-17-fatty acid esters of estradiol are extremely potent and unusually long-lived estrogens. Thus, it appeared that increased potency would be a logical consequence of the esterification of all of the active steroid hormones. To test this hypothesis we measured the effect of an ester of corticosterone, corticosterone-21-stearate, on the induction of tyrosine aminotransferase in adrenalectomized rats. Surprisingly, while the ester is active compared to the unesterified corticoid corticosterone, there was no difference in either the magnitude or the duration of the induction of this enzyme. To determine whether the C-21-steroidal ester could itself induce this gluconeogenic response, we tested corticosterone-21-oleate and corticosterone-21-stearate as competitors for the binding of [3H] dexamethasone to the glucocorticoid receptor in rat liver cytosol. Both were poor ligands, with binding affinities of about 4% and more than 1%, respectively, compared to corticosterone. From these results, it is doubtful that the esters could act directly in vivo without prior cleavage of the fatty acid. We measured the rate of hydrolysis of corticosterone-21-stearate and estradiol-17-stearate by rat liver esterases. Corticosterone-21-stearate is hydrolyzed at a much greater rate (10- to 25-fold) than estradiol-17-stearate. Consequently, the difference in both potency and duration of response between the ester of the corticoid and that of the estrogen can be explained by the very rapid rate of conversion of the former into the unesterified form. Since the esterification of the corticoids appears not to be related to an increased biological half-life, as it is in the estrogens, the question remains as to the physiological role that they might play.

16 Alpha-[125I]iodo-11 beta-methoxy-17 beta-estradiol: a radiochemical probe for estrogen-sensitive tissues.

We have synthesized an analog of 16 alpha-iodoestradiol, 11 beta-methoxy-16 alpha-iodo-estra-1,3,5-(10)triene-3,17 beta-diol (16 alpha-iodo-11 beta-methoxyestradiol), as a potential radiopharmaceutical for the in vivo imaging of estrogen-sensitive tissues. This steroid was synthesized labeled with 125I by halogen exchange of the stable intermediate 11 beta-methoxy-16 beta-bromo-17 beta-estradiol with Na125I. The halogen exchange reaction produces the radioiodinated steroid with a 65-80% yield in 3 h. This rapid synthesis and purification of the 125I-labeled estrogen permits a similar synthesis with 123I, a radioisotope with excellent properties for imaging. The 11-methoxy analog is a highly potent estrogen that binds to the estrogen receptor with an affinity equal to that of estradiol. In vivo, 11 beta-methoxy-16 alpha-[125I]iodoestradiol concentrates in an estrogen receptor-dependent manner in the uterus, producing remarkably sustained and much higher uterus to blood ratios than 16 alpha-[125I]iodoestradiol. Thus, this radiosteroid shows great promise, both as a research probe of the estrogen receptor and as a clinical tool for the imaging of estrogen-responsive tumors.

Measurement of estradiol-17-fatty acid esters in human tissues.

We have developed a gas chromatographic/mass spectral method for the sensitive and reproducible measurement of estradiol-17-fatty acid esters in human tissues and blood. To provide an internal standard for quantification, a trideuterated analog of a representative estradiol ester is added to the tissues. Estradiol (E2) released from the nonpolar ester fraction by alkaline hydrolysis is derivatized to form the ditrimethylsilyl ether and then analyzed by gas chromatographic/mass spectral, monitoring the molecular ions mass per U charge of the ditrimethylsilyl derivative of E2 and [2H3]E2. There are low but detectable levels of E2 ester in the blood of cycling females; there are none in urine. While the E2 ester is present in breast cyst fluid, its concentration, 77-140 pmol/L, is considerably less than E2, 110-2,863 pmol/L. But there is a large amount of E2 ester in fat. In premenopausal women the average E2 ester in fat (sc and omental) is 957 +/- 283 38 fmol/g (SEM); in women who are menopausal less than 12 yr, the E2 ester in fat is 669 +/- 158 fmol/g; in women who are menopausal at least 15 yr, the fat level is 399 +/- 146 fmol/g. Muscle from the same women have lower concentrations of the ester; in 8 out of 12 muscle specimens it was not detectable. The E2 esters are extremely potent estrogens. Although they are hormonally active they require enzymatic hydrolysis to exert their hormonal action. These studies show that these long chain esters of E2 are sequestered in fatty tissues, wherein they represent a protected store of preformed hormone. Under the proper stimulation, adipose tissue can activate the estrogenic signal through the action of hormonally sensitive esterases. Thus, through signaling between estrogen sensitive tissues and neighboring fat cells, a local paracrine loop may exist.

Metabolism of estradiol fatty acid esters in man.

Fatty acid esters of estradiol coupled at the C-17 position are naturally occurring metabolites of estradiol (E2). They have been recovered after incubation of E2 with human tissue and identified in human plasma. We investigated the primary metabolic transformation of E2, namely C-17 oxidation, of two representative fatty acid esters, in five normal subjects (four men and one woman), aged 25-55 yr, using a radiometric method. The transfer of tritium from the C-17 alpha position to tritium water after iv injection of free E2 was compared to that of E2-17 beta-stearate and E2-17 beta-arachidonate. Both esters were oxidized at the C-17 position to a greater extent than was free E2. In addition, the oxidation of the E2 fatty acid esters proceeded more slowly. Thus, the time necessary to reach half the maximal extent of reaction ranged from 30-45 min for the three E2-17 beta-arachidonate studies and from 2.5-4 h for the five E2-17 beta-stearate studies, while that for free E2 was less than 15 min in each instance. The disappearance of intact E2-17 beta-stearate from plasma after bolus injection was studied in two subjects (one of the five above and one additional subject). The t1/2 values calculated for the 0-60 min period were 24 and 16 min. The rate of disappearance E2-17 beta-stearate was slower than that of E2. The biological activity of E2 esters is thought to reside in their ability to be converted, by hydrolysis, to free E2. The E2 esters must undergo hydrolysis before the oxidation of free E2 to estrone can proceed. Since the oxidation reaction is extremely rapid, studying the rate of oxidation after injection of the E2 fatty acid esters provides an index of their in vivo hydrolysis and, thus, a measure of their subsequent biological activity as the free hormone. The unsaturated arachidonate ester of E2 was oxidized at a faster rate than the saturated stearate ester; this implies that the hydrolysis of the arachidonate ester was faster. Evidence of continuing oxidation of E2-17 beta-stearate at times well after its level in plasma markedly decreased indicates that the ester is removed from the circulation before its subsequent hydrolysis and oxidation. We conclude that in man, the C-17 oxidation of both E2-17 beta-arachidonate and E2-17 beta-stearate proceeds more slowly but to a greater extent than that of the free steroid.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning and characterization of Chinese hamster p53 cDNA.

We have cloned and sequenced Chinese hamster p53 cDNA and have compared the p53 sequence in different Chinese hamster cell lines to several relevant phenotypes. Our results indicate that a mutation in CHO cells that changes Thr211 to Lys211 abrogates the ability to arrest in G1 and apparently renders cells capable of amplifying DNA. However, this mutation has no effect on the G2 checkpoint or on acute down-regulation of DNA replication after a radiation challenge.

The synthesis of 11 beta-methoxy-[16 alpha-123I] iodoestradiol and its interaction with the estrogen receptor in vivo and in vitro.

In order to produce an estrogen receptor mediated imaging agent, we have synthesized 11 beta-methoxy-16 alpha-iodoestradiol labeled with 123I, and have studied its interaction with the estrogen receptor and its distribution in rats and rabbits. This 123I-labeled steroid, 11 beta-methoxy-16 alpha-[123I]iodoestradiol, binds with high affinity, Ka = 6 x 10(9) M-1, and specificity to the estrogen receptor in uterine cytosol. When tested in vivo, this radiolabeled steroid concentrates by a receptor mediated mechanism, in the estrogen target tissue, the uterus, producing very high target to nontarget tissue ratios. The results of these experiments indicate that 11 beta-methoxy-16 alpha-[123I]iodoestradiol may be a useful imaging agent for clinically monitoring and detecting estrogen receptor containing tumors.

Effects of two progestin-only contraceptives, Depo-Provera and Norplant-II, on the vaginal epithelium of rhesus monkeys.

The objective of this study was to determine whether progestin-only contraceptives induce thinning of the vaginal epithelium in nonhuman primates. Eight intact rhesus monkeys (four per group) were treated with either a single intramuscular injection of 30 mg of Depo-Provera or a subcutaneous insertion of Norplant-II (2 x 75 mg rods; day 0). Norplant-II rods were removed 90 days after insertion. Vaginal biopsies were obtained during a pretreatment menstrual cycle and following treatment on days 10, 30, 60, 118, and 146. Formalin-fixed vaginal biopsies were evaluated for epithelial thickness and the degree of keratinization. The circulating levels of estradiol, progesterone, medroxyprogesterone acetate (MPA), or levonorgestrel (LNG) were monitored throughout the study by specific radioimmunoassays. Circulating levels of estradiol and progesterone confirmed the stage of the menstrual cycle in which pretreatment biopsies were obtained. Following treatment with Depo-Provera, serum levels of MPA increased to 2.3 +/- 0.6 ng/ml (x +/- SE, n = 4) within 24 hr. Serum levels of MPA were maximal on day 14 (5.5 +/- 0.9 ng/ml), dropped below 1 ng/ml by day 50, and were nondetectable by day 70. Circulating levels of LNG were elevated 24 hr after insertion of Norplant-II (5.8 +/- 3.0 ng/ml), peaked on day 2 (7.6 +/- 4.2 ng/ml), remained between 1.4 and 6.2 ng/ml from days 14 to 90, and were nondetectable by day 118, the first serum sample after removal of Norplant-II. There were no significant differences (p > 0.05) in the epithelial thickness (microm), number of epithelial cell layers, or type of epithelium present in vaginal biopsies obtained during the follicular or luteal phases of the pretreatment menstrual cycle. Conversely, a pronounced effect of progestin treatment was observed on the vaginal epithelium. There were no significant differences (p > 0.05) between the two progestin treatment groups, but a significant effect (p < 0.05) over time was observed (two-way ANOVA). Compared with pretreatment menstrual cycle controls, the vaginal epithelial thickness was decreased (p < 0.05) by day 30 or 60 following Norplant-II insertion or Depo-Provera injection, respectively. The number of epithelial cell layers was also decreased (p < 0.05) on days 30 and/or 60 in progestin-treated monkeys compared with pretreatment control cycles. Following removal of Norplant-II or metabolic excretion of MPA, the vaginal epithellium regenerated and the thickness was no longer different (p > 0.05) from the pretreatment control cycle. These data demonstrate that progestin-only contraceptives induced thinning of the vaginal epithelium in rhesus monkeys, and this effect was rapidly reversible following physical or metabolic removal of the progestin.

The radiation-induced S-phase checkpoint is independent of CDKN1A.

We recently demonstrated that, in response to radiation, replication is down-regulated at the level of individual origins throughout S phase of the cell cycle. Since several in vitro studies demonstrate that CDKN1A (formerly known as p21) down-regulates replication by inhibiting PCNA, and since CDKN1A can retard progression of cells through S phase in vivo, the question arises whether CDKN1A is involved in the S-phase damage-sensing pathway. In the present study we analyzed the effect of ionizing radiation on CDKN1A+/+ and CDKN1A-/- cells derived from the HCT 116 cell line. Neither progression of cells through S phase nor survival after exposure to ionizing radiation is influenced by CDKN1A status in either synchronous or asynchronous cells. These results establish that CDKN1A is not necessary for the acute S-phase damage-sensing pathway that functions to prevent firing of replication origins during S phase.

Steroidal fatty acid esters.

Several years ago we discovered an unexpected family of steroidal metabolites, steroidal fatty acid esters. We found that fatty acid esters of 5-ene-3 beta-hydroxysteroids, pregnenolone and dehydroisoandrosterone are present in the adrenal. Subsequently, others have shown the existence of these non-polar 5-ene-3 beta-hydroxysteroidal esters in blood, brain and ovaries. Currently, almost every family of steroid hormone is known to occur in esterified form. We have studied the esters of the estrogens and glucocorticoids in some detail, and have found that these two steroidal families are esterified by separate enzymes. In a biosynthetic experiment performed simultaneously with estradiol and corticosterone, we established that the fatty acid composition of the steroidal esters is quite different. The corticoid is composed predominantly of one fatty acid, oleate, while the estradiol esters are extremely heterogeneous. Our studies have demonstrated that the estrogens are extremely long-lived hormones, that they are protected by the fatty acid from metabolism. They are extremely potent estrogens, with prolonged activity. Esterification appears to be the only form of metabolism that does not deactivate the biological effects of estradiol. We have demonstrated the biosynthesis of fatty acid esters of estriol, monoesters at both C-16 alpha and C-17 beta. They too are very potent estrogens. These fatty acid esters of the estrogens are the endogenous analogs of estrogen esters, like benzoate, cypionate, etc., which have been used for decades, pharmacologically because of their prolonged therapeutic potency. We have found that the estradiol esters are located predominantly in hydrophobic tissues, such as fat. Sequestered in these tissues, they are an obvious reservoir of estrogenic reserve, requiring only an esterase for activation. To the contrary the biological activity of the fatty acid esters of the glucocorticoid, corticosterone, is not different from that of its free parent steroid. We have shown that the rapid kinetics of its induction of gluconeogenic responses is caused by its labile C-21 ester which is rapidly hydrolyzed by esterase enzymes. While it appears that the physiological role of the estrogen esters may be related to their long-lived hormonal activity, the role of the other families of steroidal esters is not yet apparent. They, and perhaps the estrogen esters as well, must serve other purposes. Indeed they may serve important biological functions beyond those which we ordinarily associate with steroid hormones.

Estrogenic action of estriol fatty acid esters.

Recent studies suggest that, estriol, like estradiol, is biosynthetically esterified with fatty acids. We have synthesized the stearate estriol, at C-16 alpha, C-17 beta and the diester, C-16 alpha,17 beta and tested these D-ring esters for their estrogenic action both in vivo and in vitro, comparing them to estradiol, estriol and estradiol-17-stearate. None of the estriol esters bind to the estrogen receptor. They are only weakly estrogenic in a microtiter plate estrogen bioassay: stimulation of alkaline phosphatase in the Ishikawa endometrial cells. However, both estriol monoesters are extremely potent estrogens when injected subcutaneously (in aqueous alcohol) into ovariectomized mice. Compared to the free steroids, they produced a dramatically increased uterine weight with a greatly prolonged duration of stimulation. The 16 alpha,17 beta-diester also induced a protracted uterotrophic response, but the stimulation of uterine weight was comparatively low. Since the esters of estradiol and estriol do not bind to the estrogen receptor, their estrogenic signal must be generated through the action of esterase enzymes. These naturally occurring esters have the potential of being extremely useful pharmacological agents for long-lived estrogenic stimulation.

A simple procedure for solubilizing 3beta-hydroxysteroid dehydrogenase from microsomes of rat adrenals and testis interstitial cells.

A simple procedure is described for solubilizing microsomal 3beta-hydroxysteroid dehydrogenase (3beta-HSD). Microsomes from rat adrenals or from testicular interstitial cells were incubated for 1 or 2 h at 0 C in a buffer containing NaCl followed by overnight storage at -20 C. Maximum solubilization of 3beta-hydroxy-5beta-androstan-17-one-HSD (androstane-3beta-HSD) was obtained by incubating adrenal microsomes with 1 M NaCl and interstitial cell microsomes with 2 M NaCl. Incubation with NaCl for 1 or 2 h resulted in maximum solubilization; incubation with NaCl for 4, 8 or 24 h did not change the amount of enzyme solubilized. From adrenal microsomes incubated with 1 M NaCl, up to 80% (105.7 millimicron/mg microsomes) of the total androstane-3beta-HSD activity was recovered in the supernatant following centrifugation at 130,000 x g for 1 h. The maximum amount of androstane-3beta-HSD solubilized from interstitial cell microsomes was 56% (29.5 millimicron/mg microsomes) at 2 M NaCl. The "solubilized" androstane-3beta-HSD was retarded when chromatographed on a Sephadex G-200 column and it did not pellet out when centrifuged at 130,000 x g for 15 h. KCL appeared to be equally effective in solubilizing androstane-3beta-HSD from microsomes. Other steroid dehydrogenase activities such as pregnanolone-HSD and 3beta-hydroxy-5alpha-androstan-17-one-HSD were also found in the 130,000 x g supernatant.

Aromatase and testosterone fatty acid esters: the search for a cryptic biosynthetic pathway to estradiol esters.

The estradiol fatty acid esters (lipoidal derivatives, LE2) are extremely potent estrogens that accumulate in fat, including fat of menopausal women. These steroidal esters are protected from metabolism and are converted to the free, biologically active steroid through the action of esterases. Previous studies have shown that biosynthetic pathways in the adrenal gland exist in which steroid fatty acid esters are substrates. This led us to determine whether a cryptic aromatase pathway exists in which testosterone esters could be converted directly into LE2. We tested a representative fatty acid ester, testosterone stearate, both as an inhibitor and as a substrate for the aromatase enzyme from human placental microsomes. This ester had neither activity. In addition, we tested [1 beta-3H]testosterone acetate as a substrate for this enzyme complex, measuring the production of 3H2O as evidence of aromatization. Although the rate of reaction was considerably slower than that of testosterone, 3H2O was produced. However, when [2, 4, 6, 7-3H]testosterone acetate was incubated and the steroidal products isolated, we found that hydrolysis of the substrate had occurred. Both [3H]-labeled testosterone and estradiol were found, and very little if any [3H]estradiol acetate was formed. Thus, we conclude that an aromatase pathway involving testosterone esters does not exist and that the sole source of LE2 is through direct esterification of estradiol.

Phase I/II trial of superselective arterial 5-FU infusion with concomitant external beam radiation for patients with either anaplastic astrocytoma or glioblastoma multiforme.

From August 1987 through September 1989, 25 patients with either anaplastic astrocytoma (8 patients) or glioblastoma multiforme (17 patients) were entered into a Phase I trial of combined intra-arterial 5-fluorouracil (5-FU) and external beam radiation therapy. The intra-arterial 5-FU was given in a superselective, supraopthalmic fashion, in escalating doses from 200 mg to 600 mg on a weekly basis during the radiotherapy. Each patient received from 1-4 courses of 5-FU. Radiotherapy consisted of 5,000 rads in 25 fractions given to partial brain fields including the mass and surrounding edema plus a 3-cm margin as defined by computed tomography scan. There were a total of 4 significant acute complications out of a total of 70 infusions. These included 3 ischemic events of which 2 were transient. In addition, 1 patient experienced a cerebral bleed. No patients developed ocular complications. Electroencephalograms (EEGs) were performed immediately before and during 21 intra-arterial infusions. Two patients developed significant EEG changes during intra-arterial infusion, and both of these patients experienced untoward reactions. The remaining patients showed no EEG changes during their infusions. The median survival for patients with glioblastoma multiforme was 15 months. We believe the toxicity of superselective intra-arterial 5-FU infusion combined with external beam radiotherapy is acceptable. This type of treatment deserves further study.