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Regular Article |
a Institute of Biology, Faculty of Sciences, University of Novi Sad, 21000 Novi Sad, Serbia
b Endocrinology and Reproduction Research Branch, NICHD, Bethesda, Maryland 20892
ABSTRACT
Polychlorinated biphenyls (PCBs) are complex mixtures of congeners that exhibit carcinogenic and toxicant activities in a variety of mammalian tissues. Here, we studied the acute in vivo and in vitro effects of a commercially used PCB product, Aroclor 1248 (A1248), a mixture of tri-, tetra-, and pentachloro congeners. Single intraperitoneal (i.p.) or bilateral intratesticular (i.t.) injections of A1248 decreased serum androgen levels in both groups 24 h after injection. Chorionic gonadotropin–stimulated androgen production by acute testicular cultures from both groups was also reduced, and progesterone production was attenuated in cultures from i.t.-treated animals. The capacity of the postmitochondrial fractions from testes of i.t.-treated animals to convert pregnenolone to progesterone and progesterone to testosterone was reduced as well. In vitro studies revealed that a 10- to 15-min exposure of postmitochondrial testicular fractions and intact interstitial cells from normal animals to A1248 in a subnanomolar concentration range was sufficient to attenuate the conversion of pregnenolone to progesterone and progesterone to testosterone. At micromolar concentrations, A1248 added in vitro also inhibited the conversion of
4-androstendione to testosterone without affecting the viability of interstitial cells. These results indicate that A1248 down-regulates the testicular androgenesis by an acute inhibition of 3ß-hydroxysteroid dehydrogenase, 17
-hydroxylase/lyase, and 17ß-hydroxysteroid dehydrogenase activities.
Frequently used industrial products, polychlorinated biphenyls (PCBs), belong to a class of environmentally persistent compounds with known carcinogenic and hepato-, immuno-, and neurotoxicant activity. The interference of PCBs with reproductive functions is also well documented. The majority of previously published studies focused on the in vivo effects of PCBs, such as fetal toxicity, developmental malformation, a decrease in reproductive ability, and multiple testicular abnormalities [1–7]. Several studies also addressed in vivo and in vitro effects of PCBs on steroidogenesis by gonadal and adrenal tissues [1, 8–13]. The mechanism of PCB-induced abnormalities in reproductive tissues and steroidogenesis is not well defined. Two major groups, the coplanar PCBs and the ortho-substituted PCBs, differ in their actions. The coplanar PCBs operate through the aromatic hydrocarbon (Ah) receptor signaling pathway in a manner comparable to that observed with 2,3,7,8-tetrachloro-
-dioxin (TCDD), polychlorinated dibenzofurans (PCDFs), and related substances [14–16]. The mono-ortho-substituted PCB congeners also bind to the cytosolic Ah-receptor, but with lower potency than coplanar congeners. [7]. The PCBs with two or more ortho-chlorines exhibit low or no affinity for the Ah-receptors [17–19] but can induce hormone- and neurotransmitter-like effects [14–16]. For example, some PCBs exhibit estrogenic and antiestrogenic actions that may contribute in altering the reproductive function [20].
Aroclors (A) are commercially used PCB mixtures, with a variable percentage of chlorine denoted by the last two numbers in their names. The frequently used compounds from this family include A1260, A1254, A1248, A1242, and A1016 [21]. In general, the actions of these compounds in endocrine and reproductive tissues have not been studied extensively. A1254, a constituent of a number of transformers' oils [22], is the most studied Aroclor. Repetitive administration of this mixture in female minks leads to morphological changes in ovaries and uteri, as well as to increased fetal mortality associated with changes in placental development [4]. Effects of A1254 on mouse oocyte, spermatozoa, in vitro fertilization, and embryo development have also been reported [23].
Another member of the group, A1248, has the following weight percentage of PCB congener distribution: less than 2% of mono- and di-, 21% of tri-, 55% of tetra-, 21% of penta-, and less than 2% of hexachlorobiphenyl [21]. Similar mixtures were frequently used in a number of industrial products, such as polymer concrete join sealant and the nonmetal components of home appliances and cars [22]. In comparison to other Aroclor mixtures, A1248 contains a significant amount of coplanar congener PCB 77 (3,3',4,4'-tetrachlorobiphenyl [21]), which should be able to activate the Ah-receptor signaling pathway. Moreover, A1248 contains mono-ortho congeners that are Ah-receptor agonists with lower potency and di-ortho congeners that could generate hormone- and neurotransmitter-like effects. Also, congener PCB 77 can act as an estrogen [24], even though coplanar Ah-receptor agonists are generally considered to be antiestrogenic [20, 25]. Thus, the potential toxic effects of A1248 and related compounds may be amplified due to multiple actions on signaling pathways. However, the toxicity of this frequently used product has not been experimentally addressed.
In this study, we investigated the possible toxic effects of A1248 on rat testicular androgenesis and the mechanism of its action. We compared intraperitoneal (i.p.) with intratesticular (i.t.) routes of A1248 administration on testicular steroidogenesis during a prolonged in vivo exposure (24 h). In addition, we examined the capacity of postmitochondrial testicular fractions from normal animals to produce steroids during a short-term in vitro exposure (10–15 min) to different concentrations of A1248. The possible cytotoxic effects of A1248 were also addressed. The results of these investigations indicate that A1248 down-regulates the testicular steroidogenesis at subnanomolar concentrations and that this action is not mediated by the Ah-receptor signaling pathway.
MATERIALS AND METHODS
Chemicals
A1248 was obtained from Supelco (Monsanto, CA). Antitestosterone-11-BSA serum #250 and antiprogesterone-11-BSA serum #337 were supplied by G. D. Niswender (Colorado State University, Fort Collins, CO). Medium 199 (M199) was purchased from GIBCO Laboratories (Gaithersburg, MD); NADPH, NADH, NAD, cytochrome c, BSA (fraction V), collagenase (type I), trypan blue, testosterone, 4-androstenedione, progesterone, and pregnenolone were obtained from Sigma (St. Louis, MO). [1,2,6,73H(N)]-testosterone and [1,2,6,73H(N)]-progesterone were obtained from New England Nuclear (Boston, MA), Dextran T70 was purchased from Pharmacia (Uppsala, Sweden). Norit A charcoal was purchased from Serva (Heidelberg, Germany) and Tris (hydroxymethyl) aminomethane from Bethesda Research Laboratories (Bethesda, MD). All other reagents were of analytical grade.
Animals, Treatments, and In Vitro Steroidogenesis
Male Wistar rats, raised under controlled environmental conditions (temperature 22 ± 2°C and 14L:10D) in our laboratory with food and water ad libitum, were used for experiments. The desired concentrations of A1248 fluids were prepared by evaporating the required amount of stock solution and dissolving it in oleum olive or saline. Rats were handled daily during a 1-wk acclimation period prior to experiments and then treated with an i.p. injection of either vehicle (oleum olive) or with A1248 (10 mg/kg body weight) or an i.t. injection of either vehicle (saline) or A1248 (25.5 µg/testis) between 0800–0830 h.
The following experiments were conducted in accordance with the principles and procedures of the NIH Guide for care and use of laboratory animals, and the Local Animal Ethical Committee of the Institute of Biology. Animals were sacrificed by decapitation 24 h after injections, trunk blood was collected, and serum samples were stored at -20°C until analyzed for testosterone + dihydrotestosterone (T+DHT) content by RIA. Testes from control and treated rats were quickly removed, decapsulated, weighted, and incubated individually in vials containing a total of 5 ml medium 199 enriched with 0.1% BSA, and 20 ng/ml hCG. The contents of the incubation vials were gassed with 95% O2-5% CO2 and incubation was carried out for 3 h at 34°C in a shaking waterbath (100 oscillations/min). Incubation medium was decanted and centrifuged for 10 min at 1500 x g and individual samples of supernatants were stored at -20°C before measurement of androgens (T+DHT) and progesterone levels by RIA.
Enzyme Activities in Postmitochondrial Testicular Fractions
To prepare the postmitochondrial fractions, testes were decapsulated and homogenized in 50 mM phosphate buffer containing 0.25 M sucrose (pH 7.4), using a glass-glass homogenizer. After centrifugation (4°C for 20 min at 1500 x g) the supernatants were mixed with dextran-coated charcoal in order to remove the endogenous steroids [26]. The samples were centrifuged at 1500 x g for 10 min, and supernatants were further centrifuged at 12 000 x g for 20 min. Protein content in postmitochondrial fractions was estimated by Bradford method [27], using BSA as a standard. To estimate the in vitro effect of A1248 on enzyme activities, the desired concentrations of A1248 was prepared by evaporating the necessary amount of stock solution, dissolving it in 0.1 M phosphate buffer, and adding it directly into the corresponding reaction mixture.
The P450c17 and 17ß-hydroxysteroid dehydrogenase (17ßHSD) activities in postmitochondrial fractions were measured as previously described [28] and estimated by the conversion of progesterone to testosterone and 4-androstenedione to testosterone, respectively. Briefly, in the final volume of 0.25 ml, the incubation solution contained saturating concentrations of steroid substrates (10 µM), 1 mM NADPH, 0.1 M phosphate buffer (pH 7.4), and 0.1 ml postmitochondrial fractions (0.35 mg proteins/tube). Mixtures were incubated for 15 min at 37°C in a shaking waterbath in 95% O2-5% CO2 atmosphere. The 3ß-hydroxysteroid dehydrogenase (3ßHSD activity) was estimated as previously described [28] through the conversion of pregnenolone to progesterone. The incubation solution, with a final volume of 2 ml, contained 25 µM pregnenolone, 135 µM NAD+, 100 µM phosphate buffer (pH 7.4), and 0.1 ml of the postmitochondrial fraction (0.22 mg proteins/tube). Mixtures were incubated for 10 min at 37°C in a shaking waterbath in an atmosphere of 95% O2-5% CO2.
The activities of P450c17 and 17ßHSD were measured in the presence of saturated concentrations of corresponding steroid substrates, whereas 3ßHSD activity was measured in the presence of subsaturating concentration of pregnenolone (the estimated Km = 8.43 ± 1.76 mM). Selected incubation times were within temporal linearity of the enzyme activities [28–30]. Enzyme reactions were initiated by the addition of 0.1-ml postmitochondrial fractions and terminated by placing the tubes in an ice-cold bath. The samples were stored at -20°C until assayed for testosterone or progesterone by RIA.
Androgen Production by Interstitial Cells
Adult male rats (around 3 mo old) were sacrificed by decapitation. Testes were quickly removed, decapsulated, and enzymatically dispersed with collagenase according to Anakawe et al. [31], with some modification [30]. Briefly, for each experiment two testes were placed in a 50-ml plastic tube containing 3 ml of collagenase solution (1.2 mg/ml collagenase and 1 mg/ml BSA in M199) and incubated at 34°C for 15 min. Following dispersion, filtration, and centrifugation steps (three times), the cell pellet was resuspended in a corresponding amount of M199 containing 0.1% BSA. The 0.2% trypan blue dye exclusion test (Sigma) was used to determine total cell counts and viable cell number, which was more than 95%.
Aliquots (106 cells/0.3 ml) of crude suspension of rat interstitial cells were added to 12 x 75-ml plastic tubes containing 2 µM of steroid substrate (progesterone or 4-androstenedione) with or without different concentrations of A1248. The desired concentrations of A1248 were prepared by evaporating the necessary amount of stock solution and dissolving it in M199-BSA. All tubes were incubated in six replicates, for 2 h in a shaking waterbath oscillating at 100 cycles/min under an atmosphere of 95% O2-5% CO2. After incubation tubes were centrifuged for 5 min at 400 x g at 4°C, and the supernatant was stored at -20°C. The pelleted cells in the tube were resuspended in M199-BSA (0.4 ml/tube) and cell viability was assessed by trypan blue exclusion test. Acute cytotoxicity was calculated according to the following equation: acute cytotoxicity = (1 - VT/VC) x 100, where VT and VC are viability (V) in the presence (VT) and absence (VC) of drug.
NADPH-P450 Reductase Activity in Postmitochondrial Fractions of Interstitial Cells
The NADPH-P450 reductase activity in postmitochondrial fractions of interstitial cells was measured as previously described [32]. Briefly, interstitial cells were resuspended in 5 ml of 17 mM Tris, 140 mM NH4Cl solution (pH 7.2) and incubated for 10 min at room temperature. This procedure eliminates red blood cells and the interference of hemoglobin. The cell pellets were washed twice with M199-BSA, and twice with 0.9% saline. Postmitochondrial fractions of interstitial cells were prepared as described above. The NADPH-P450 reductase activities were measured as the change in absorbancy at 550 nm. The cuvette (final volume 0.6 ml) contained 0.2 mM NADPH, 1 mM KCN, 30 µM cytochrome c, postmitochondrial fraction (0.15 mg proteins/cuvette) in 0.1 M phosphate buffer and without (control) or with increasing doses of A1248. The reactions were initiated by the addition of NADPH that was omitted from the blanks [32, 33]. The millimolar extinction difference between reduced and oxidized cytochrome c was 21.2 at 550 nm.
Hormone Assays
Androgens and progesterone levels in serum and incubation medium were estimated by RIA. Each experiment was run in a single assay. Precision of androgen assay was 6 pg/tube; intra- and interassay coefficients of variation were 5.8% and 7.5%, respectively. The antitestosterone serum used in RIA shows a high cross-reactivity with DHT and assay values for androgen production are referred to as T+DHT concentrations. For estimation of androgen production in vitro samples were diluted 152 times and in such conditions that there was no cross-reactivity of progesterone and 4-androstenedione with antitestosterone serum. Precision of the progesterone assay was 6 pg/tube and intra-assay and interassay coefficients of variation were 6.8% and 10.7%, respectively. Because antiprogesterone serum used in RIA assay for progesterone cross-reacts with pregnenolone with a relative binding affinity of 1.4%, additional tubes without postmitochondrial fractions and with pregnenolone were also included, and progesterone production was calculated after subtraction of these blanks for pregnenolone.
To assess the possibility of cross-reactivity between A1248 and antiprogesterone and antitestosterone sera used in our RIA assay, tubes containing 0.1 M phosphate buffer, corresponding steroid substrate, and increasing concentrations of A1248 without postmitochondrial fractions were also incubated. A1248 did not significantly cross-react with antitestosterone serum (between 0.1% and 1%, depending on added concentration). The cross-reactivity between antiprogesterone serum and A1248 was below 1%. The T+DHT and progesterone productions were calculated after subtraction of corresponding blanks for A1248.
Statistical Analysis
All results were expressed as mean ± SEM. Statistical analysis for data shown in Figures 1 and 2 was done by Student's t-test and Mann-Whitney test, with *P < 0.05 or higher in both tests. Data shown in Figure 3 and Tables 1 and 2 were analyzed by ANOVA, and posthoc comparisons between means were made by Dunnett's test.
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RESULTS
Both i.p. (10 mg/kg BW) and bilateral i.t. (25.5 µg/tesis) injections of A1248 were associated with a dramatic decrease in serum T+DHT levels 24 h after treatment (Fig. 1A). In addition, hCG (20 ng)-induced androgen production in decapsulated testes, incubated for 3 h immediately after the animals were sacrificed, was significantly inhibited by both treatments (Fig. 1B). Progesterone production by the same cultures was also inhibited but only in tissues from i.t.-injected animals (Fig. 1C). Furthermore, the ability of postmitochondrial testicular fractions from treated animals to convert pregnenolone to progesterone was reduced by 37%, while the conversion of progesterone to testosterone was reduced by 50% (Fig. 2). These results suggest that A1248 exhibits an inhibitory effect on serum androgens and on hCG-stimulated and progesterone-derived T+DHT production independent of the route of administration.
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A1248 also significantly decreased the conversion of pregnenolone to progesterone when added directly to the postmitochondrial fractions of normal rats, indicating an inhibitory effect of this compound on 3ßHSD activity. The conversion of progesterone to T+DHT was also affected by A1248. As shown in Table 1, both conversions were inhibited at (sub)nanomolar concentrations. Addition of A1248 also decreased the activity of NADPH-P450 reductase in all concentrations tested (Table 2). The activity of this enzyme, however, remained within 90% of the control. Thus, inhibition of NADPH-P450 reductase alone cannot explain the attenuated conversion of progesterone to T+DHT. Because A1248 did not have an effect on the conversion of 4-androstenedione to T+DHT in 0.1 nM to 50 µM concentration range (Table 1), these results further indicate that the major action of A1248 on the conversion of progesterone to T+DHT occurs through inhibition of P450c17 activity.
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We have also analyzed in vitro effects of short-term exposure of A1248 on intact interstitial cells from normal animals. Figure 3, upper line, illustrates that A1248 did not affect the viability of isolated interstitial testicular cells for all concentrations studied. However, as in postmitochondrial testicular fractions, conversion of progesterone to T+DHT was also inhibited in a concentration-dependent manner in intact cells, with an estimated EC50 in a subnanomolar concentration range. Furthermore, this inhibition was specific to P450c17, because 17ßHSD activity was not inhibited, as estimated by the lack of effects of A1248 on the conversion of 4-androstenedione to T+DHT in 0.1 nM to 5 µM concentration range (Fig. 3).
Finally, our results indicate that conversion of progesterone to T+DHT in interstitial cells was inhibited in a biphasic manner. In addition to the above-described decrease at subnanomolar to nanomolar concentrations, an additional inhibition was observed at micromolar concentrations (Fig. 3, bottom line). This inhibition paralleled a significant reduction in the conversion of 4-androstenedione to T+DHT at 10 µM concentration in intact cells (Fig. 3, middle line) and postmitochondrial fractions containing 10 µM A1248 (Table 1). These results suggest that 17ßHSD is also inhibited by A1248 but only at micromolar concentrations.
DISCUSSION
Our study focuses on the effects of A1248, a mixture of different PCBs, on rat testicular steroidogenesis. The results indicate that A1248 inhibits testicular steroidogenesis when administrated in vivo and when added in vitro. This inhibition was rapid and occurred at subnanomolar to nanomolar concentrations of A1248. When expressed in toxicological terms, concentrations of A1248 used in our in vitro studies were in the range of 0.028 ppb to 28 ppm. General levels of PCBs in soil and sediment are in the range of parts per billion, while in water in subparts per trillion [22]. Polychlorinated biphenyls are found consistently in many environmental matrices, including marine plants and animals, freshwater fish, mammals, wildlife, soils, air, and water. The estimated contents of PCBs in samples of carp and pike taken from the European rivers Tisa, Sava, and Danube were in the range of 9 to 25 ppb and 11 to 37 ppb, respectively. A higher content of PCBs in pike compared to carp is in accordance with bioaccumulation and bioconcentration through food-chains [34]. The congeners present in A1248 were also found in samples of other fishes [25]. Because the allowed concentrations of PCBs in food samples are in the 0.3–5-ppm range [22], the sensitivity of testicular androgenesis to A1248 in the subnanomolar to nanomolar concentration ranges is of potential importance for environmental contamination and altered reproductive function.
The literature on the actions of PCBs on steroidogenesis is limited and focuses on the conversion of progesterone to steroid products. For example, A1254 did not induce any change in the activity of P450c17 in the microsomal fraction of guinea pig testes, either in pretreated animals or when the compound was added directly to the microsomal mixture. On the other hand, P450c21 activity in adrenal glands was inhibited by A1254, reflected in a decrease in the production of 11-deoxycortisol and 11-deoxycorticosterone [11]. In rat interstitial cell preparations, however, PCB mixtures of ortho-isomers and congeners with high chlorine content decreased the activity of P450c17, leading to attenuation in progesterone-supported androgen production [13].
Here, we measured the activities of several steroidogenic enzymes by following the conversion of corresponding substrate to a specific product. For 3ßHSD activity, conversion of pregnenolone to progesterone was estimated by measuring progesterone levels after a 10-min incubation of postmitochondrial testicular fractions with pregnenolone and NAD+. Because the additional conversion of progesterone to hydroxylated products (16-hydroxyprogesterone and 17-hydroxyprogesterone, the latter being further metabolized to 4-androstenedione and testosterone) requires the presence of NADPH, in our experimental setup such conversion was stopped by not adding NADPH. It has been shown previously [35] that the levels of testosterone in the incubation medium of postmitochondrial fractions from normal rats were less than 0.45 ng/min/mg protein. In the same samples, the levels of progesterone after a 10-min conversion of pregnenolone were about 350–650 ng/min/mg protein. Therefore, changes in the conversion of pregnenolone to progesterone should reflect changes in the activity of 3ßHSD.
Our results also show a decreased conversion of progesterone to testosterone in postmitochondrial fractions of A1248-treated rats, as well as the fractions with in vitro addition of the compound. The effects of A1248 on this steroid conversion are complex and involved three enzymatic steps. A decreased conversion of progesterone to testosterone at sub- to nanomolar concentrations of A1248 accompanied by unchanged conversion rate of 4-androstenedione to testosterone could be taken as a measure of decreased P450c17 activity. Because NADPH-P450 reductase is an obligatory redox partner of P450c17 enzyme by providing it with electrons [36, 37], inhibition of NADPH-P450 reductase activity by nanomolar A1248 should also be accounted for by the observed attenuation of P450c17 activity. Furthermore, results from intact interstitial cells, as well as from postmitochondrial fractions, have shown that 17ßHSD is also inhibited by A1248 but only at a micromolar concentration range.
Despite documenting the effects of A1248 on in vivo and in vitro androgenesis, our results did not significantly enhance our understanding of this mixture's mechanisms of action. Within the Aroclors, A1248 contains the coplanar PCBs, namely congener 77 (3,3',4,4'-tetrachlorobiphenyl) [21], which is of utmost importance in the evaluation of its toxicity through the Ah-receptor pathway. For example, a single dose of TCDD (100 µg/kg), a compound that binds to the Ah-receptor, impaired hCG-stimulated testosterone secretion from rat testes, presumably through a reduction in the activity of cytochrome P450scc and/or impairment of the multistep processes responsible for mobilizing cholesterol [38]. Additionaly, TCDD affects estradiol secretion by depleting 4-androstenedione precursors and increasing the apoptotic cell death of human luteinized granulosa cells, in a dose- and time-dependent fashion [39]. Coplanar PCB congeners and mono-ortho-PCB congeners also exhibited TCDD-like activity by activating the Ah-receptor pathway [7, 14].
Although the coplanar and mono-ortho-PCBs from A1248 can operate through an Ah-receptor signaling pathway, it is unlikely that the activation of these receptors by this mixture accounted for the down-regulation of androgenesis in our experiments. A1248 not only inhibited serum androgen levels 24 h after i.p. and i.t. administration and hCG-stimulated testosterone secretion in testicular cultures from these animals but also when it was only added in vitro for 10–15 min, the latter being incompatible with the activation of Ah-receptors. Furthermore, the interference of A1248 on steroidogenesis in testicular tissue cannot be explained by the cytotoxicity of this mixture, which should affect all steps in the process. A1248 did not have an effect on the viability of cells in times during which over 60% inhibition of progesterone-supported androgen production was observed (Fig. 3).
The acute effects of A1248 and its selectivity in inhibiting the conversion of pregnenolone to progesterone and progesterone to testosterone are actions more consistent with a hormone-like effect. This hypothesis is in line with observations that certain PCBs accumulate in the pituitary tissue and that A1242 enhances responsiveness of cultured anterior pituitary cells to gonadotropin-releasing hormone in a manner comparable to that observed in estradiol-treated pituitary cultures [20]. We speculate that A1248 mimics the feedback action of steroids on testicular androgenesis, a hypothesis that needs further experimental support. In line with this, testosterone is capable of autoregulating its biosynthesis through the rapid, specific, and reversible inhibition of 17-hydroxylase activity [40]. Also, the possibility that residual intracellular messengers present in the microsomal fraction mediate the action of A1248 has to be addressed.
It is well established that the metabolism of PCBs results in the formation of various derivatives, including hydroxylated PCBs, dihydrodiols and catechols, phenolic conjugates, gluthatione conjugates, and methyl-sulfonyl metabolites, many of which are toxic themselves (for references see Moore et al. [41]). However, i.p. treatment with A1248 resulted in effects similar to i.t. treatment. The only exception is the lack of an effect on hCG-stimulated progesterone production in testicular cultures in i.p.-treated animals. This could indicate more intensive metabolism of PCBs in the liver of i.p.-treated animals that may reduce the circulating levels of active ingredient below the threshold needed for the inhibition of 3ßHSD but not P450c17.
In summary, this study demonstrates that A1248 induces an acute inhibition of rat testicular androgenesis after in vivo and in vitro applications. The interference of A1248 with rat testicular androgenesis is complex but specific. The enzyme most sensitive to A1248 was P450c17, which was inhibited after i.t. and i.p. applications, as well as through in vitro application to postmitochondrial testicular fractions from normal animals and isolated interstitial cells. The activity of 3ßHSD was also inhibited by A1248 in vitro, as well as in cultures from i.t.- but not i.p.-treated animals. A partial inhibition of NADPH-P450 reductase was also observed after in vitro addition of A1248. Finally, 17ßHSD was inhibited, but only at micromolar concentrations, an action that was not relevant to the observed in vivo effects on serum androgens. These findings provide a rationale for a number of observations on the antigonadal actions of PCBs in males.
ACKNOWLEDGMENTS
We are grateful to Dr. Gordon D. Niswender for the supply of antiserum.
FOOTNOTES
First decision: 18 November 1999.
1 A grant from the Serbia Research Association supported this research. 
2 Correspondence: R.Z. Kovacevic, ERRB, NICHD, NIH, Bldg. 49, Room 6A-36, 49 Convent Drive, Bethesda, MD 20892-4510. FAX: 301 594 7031; radmilak{at}unsim.ns.ac.yu
Accepted: January 28, 2000.
Received: October 19, 1999.
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R. J. Markelewicz Jr., S. J. Hall, and K. Boekelheide 2,5-Hexanedione and Carbendazim Coexposure Synergistically Disrupts Rat Spermatogenesis Despite Opposing Molecular Effects on Microtubules Toxicol. Sci., July 1, 2004; 80(1): 92 - 100. [Abstract] [Full Text] [PDF]
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