Timothy Schrader

Effects of Aroclors and individual PCB congeners on activation of the human androgen receptor in vitro

To investigate possible interactions between the human androgen receptor and PCBs in vitro, we have used a previously characterized human androgen receptor reporter gene assay in which PC-3 LUC(AR+) cells respond to 5alpha-dihydrotestosterone (DHT, 50 pM) with enhanced luciferase activity. The effects of Aroclors, commercial mixtures of PCBs, or polychlorinated terphenyls (PCTs) (0, 0.1, 1.0, and 10.0 microM) on luciferase activity in PC-3 LUC(AR+) cells were determined after exposure for 18 h in the presence and absence of DHT (50 pM). In the absence of DHT, none of the Aroclors induced luciferase activity but, in the presence of DHT (50 pM), Aroclors 1016, 1221, 1232, 1242, 1248, 1254, 1260, 5432, and 5442 acted antagonistically at concentrations that did not affect cell viability. Aroclor 5460 was without effect. Similarly, when PCBs found as human milk contaminants were assessed as individual congeners (each at 1 microM, where no cytotoxic effects were observed), none activated luciferase expression in the absence of DHT but PCBs 49, 66, 74, 105, and 118 completely antagonized the stimulation by DHT (50 pM) and PCBs 138, 153, and 156 were less effective antagonists, reducing the DHT stimulation by about 50%. Thus, 30% (by weight) of the PCBs in human milk are androgen antagonists (PCBs 66, 74, 105, and 118) and a further 25% are partial antagonists (PCBs 138, 153, and 156). A proportionally representative mixture of PCBs that contaminate human milk also caused the DHT-mediated activation of luciferase activity in PC-3 LUC(AR+) cells to be reduced by more than 50%.

Background bisphenol A in experimental materials and its implication to low-dose in vitro study.

In vitro low-dose studies are important to understand the mechanisms of bisphenol A (BPA) action. BPA doses used in current in vitro studies varied considerably, and doses as low as 10(-15)M have been reported. The actual doses of BPA used in the in vitro low-dose studies were rarely checked analytically, and the background BPA levels in experimental materials, which will determine the lowest BPA dose to be used, should be investigated or considered. In this study, the background BPA levels in various materials typically used in in vitro low-dose studies for BPA were investigated. Background BPA levels from the use of disposable pipettes and pipette tips were low (<0.20 ng mL(-1) or 0.88 nM). BPA was also detected in several commercial buffer solutions at levels close to the method limit of quantification (LOQ) (0.02 ng mL(-1); 0.088 nM). However, BPA was detected in all cell culture media obtained from various sources at levels ranging from 0.080 to 4.26 ng mL(-1) (or 0.35 to 19 nM) with an average of 0.83 ng mL(-1) (3.5 nM). We suggest that culture media used for low-dose BPA studies should be analysed for background BPA levels prior to use, and the medium with the lowest BPA levels should be used.

Effects of chronic exposure to octylphenol on the male rat reproductive system.

p-tert-Octylphenol (OP) is a degradation product of alkylphenol ethoxylates. OP is an endocrine disruptor known to bind to the estrogen receptor; however, effects on males are controversial. The objective of this study was to evaluate the effects of chronic exposure to OP on male reproduction. Adult Sprague-Dawley rats were administered OP for 60 d, representing 1.5 cycles of spermatogenesis. Experimental groups included a vehicle control, and three doses of OP (25, 50, or 125 mg/kg body weight [bw]) administered daily by gavage. There was a significant decrease in body weight in the 125-mg/kg group after 60 d of treatment. Both testicular and epididymal weights and histology were not altered by treatment with OP at any of the doses administered. There were no marked differences in cauda epididymal sperm counts at any doses; however, total percent sperm motility was significantly lower in rats exposed to the intermediate dose (50 mg/kg bw). There was an increase in percent static sperm cells in all OP-treated groups, with the intermediate dose (50 mg/kg) displaying a significantly higher proportion of static cells relative to untreated controls. Caput epididymal sperm motility was unaltered by OP treatment. Gene expression profiles of testes from control and high-dose-exposed rats indicate that 14 genes were modulated by at least twofold, although these changes were not statistically significant. Taken together, results from this study indicate that OP treatment of adult rats does not appear to exert major effects on male reproductive endpoints at relevant environmental exposure doses.

Oral p-Tert-Octylphenol Exposures Induce Minimal Toxic or Estrogenic Effects in Adult Female Sprague-Dawley Rats

Contamination of the environment with endocrine-disrupting chemicals (EDC) has raised concerns about potential health hazards for humans and wildlife. Human and wildlife exposure to one such ubiquitous chemical, p-tert-octylphenol (OP), are likely, due to its persistence in the environment and its presence in food, water, and items of daily use. OP is reported to bind to the estrogen receptor (ER) and alter expression of estrogen-responsive genes. Detrimental effects of OP exposures on the reproductive system have been observed in most, but not all, in vivo experiments. This study examined estrogenic effects of oral exposures of adult female rats to OP. In vitro, OP bound weakly to human ER and a co-activator protein, and accelerated proliferation of MCF-7 cells. Adult Sprague-Dawley rats were given OP by gavage daily for 35 d (25, 50, or 125 mg/kg/d). Body and organ weights and ovarian follicle populations were not significantly altered in OP-exposed adult rats, despite detectable levels of OP in reproductive organs. The estrous cycle of rats was slightly altered, but there were no significant estrogen-like changes in histomorphology or gene expression of the uterus. Prepubertal rats given 125 or 250 mg/kg OP by gavage for 3 d had reduced body weight compared to vehicle-exposed rats but failed to show any uterotrophic response, although 17α-ethinyl estradiol (EE, 10μg/kg/d, ip) induced a threefold increase in uterine weight. Overall, results suggest that toxicity will occur before estrogenic effects with oral exposures to OP. Relevant environmental exposures likely pose little risk for estrogenic effects.

Toxicokinetics of p-tert-octylphenol in male and female Sprague-Dawley rats after intravenous, oral, or subcutaneous exposures.

This study was undertaken to characterize the toxicokinetics of p-tert-octylphenol (OP), a weak estrogenic compound, in male and female rats. Male and female Sprague-Dawley rats were given a single dose of OP either by oral gavage (50, 125 or 250 mg/kg), by intravenous (iv) injection (2, 4, or 8 mg/kg), or by subcutaneous (sc) injection (125 mg/kg). In a repeated dosing experiment, rats were given OP (oral) daily (25, 50, or 125 mg/kg) for 35 d (female) or 60 d (male). Blood and tissue samples were collected and analyzed for OP content using gas chromatography with detection by mass spectrometry. Blood OP concentrations were generally higher in female than male rats following a single oral or sc administration but were similar following a single iv injection. Tissue OP concentrations were also higher in female than male rats following oral exposure, consistent with the faster metabolism of OP observed in male rat liver microsomes. After subchronic administration, blood OP concentrations were higher at the end of exposure for female (33 d) (2.26-fold, not significant) and male (57 d) (3.47-fold) rats than single dosing but there was no change in the tissue OP concentrations. Gender differences in tissue OP concentrations may contribute, in part, to gender differences in the toxicity of OP in rats. The fact that OP was found in all reproductive tissues confirms its potential for direct endocrine-like effects.