Effects of beta-estradiol and bisphenol A on heat shock protein levels and localization in the mouse uterus are antagonized by the antiestrogen ICI 182,780.

Bisphenol A (BPA) exhibits many estrogen-like effects in the rodent uterus, but not all of these can be attenuated by antiestrogens. This suggests the involvement of alternate pathways of BPA action that do not involve the estrogen receptor (ER). An examination of the in vivo effects of BPA on uterine gene expression and protein levels should contribute to an understanding of its mechanism of action. In this study we examined the dose-related effects of BPA on levels of a suite of heat shock proteins (hsps) and on the localization of hsp90alpha, a chaperone of the ER, in uteri of ovariectomized B6C3F1 mice and compared these effects with those of beta-estradiol (E2). The antiestrogen ICI 182,780 (ICI) was co-administered with BPA or E2 in order to examine the potential role of the ER. BPA, although less potent than E2, increased hsp90alpha and grp94 to similar levels, but was much less effective than E2 in increasing levels of hsp72. Treatment with 100 mg BPA/kg/day or 2 microg E2/kg/day increased hsp90alpha to 300% of control levels and altered its tissue expression pattern. In uteri of corn oil (control)-treated mice, hsp90alpha predominantly localized in the cytoplasm and nuclei of epithelial cells. Upon treatment with BPA or E2 there was increased intensity of staining in the stroma and myometrium, and in the epithelium hsp90alpha was localized almost exclusively in the cytoplasm. The effects of BPA or E2 on hsp levels and hsp90alpha localization were attenuated by ICI. These results suggest an involvement of the ER in BPA- and E2-induced increases in uterine levels of hsp90alpha, grp94, and hsp72, and localization of hsp90alpha.

Bisphenol A-induced increase in uterine weight and alterations in uterine morphology in ovariectomized B6C3F1 mice: role of the estrogen receptor.

The ability of the environmental xenoestrogen bisphenol A (BPA) to increase uterine wet weight in the rodent remains controversial, and few studies have previously examined the effects of BPA on uterine morphology. Furthermore, it is not known whether BPA-induced uterotrophic effects are, similarly to beta-estradiol (E(2)), mediated through the estrogen receptor (ER). In this study, we compared the effects of BPA on uterine wet weight and morphology to those of E(2) in the B6C3F1 ovariectomized mouse. To examine whether these effects were mediated through the ER, the antiestrogen ICI 182, 780 (ICI) was co-administered with BPA or E(2). We report that subcutaneous administration of BPA at doses between 0.8 and 8 mg/day over 4 days significantly increased mean uterine wet weights above those of vehicle (corn oil)-treated mice. The uterine weight data suggest that BPA acts as a partial agonist with an EC(50) of 0.72 mg/day compared to 19.4 ng/day for E(2). BPA (2 mg/day) and E(2) (40 ng/day) induced a significant increase in luminal epithelial height and in the thickness of both the stromal and myometrial layers of the uterus. The effects of 40 ng E(2)/day on all endpoints studied were reversed by 20 microg ICI/day. ICI at 200, but not 20 microg/day, was able to reverse the BPA (2 mg/day)-induced increase in both uterine wet weight and luminal epithelial height. ICI alone at 200 microg/day stimulated an increase in thickness of both the stroma and myometrium and did not reverse the effects of BPA (2 mg/day) on these layers. These results suggest that the BPA-induced increase in uterine wet weight and in luminal epithelial height in the ovariectomized B6C3F1 mouse are mediated by the ER.

Stability of drugs of abuse in urine samples stored at -20 degrees C.

Isolated studies of the stability of individual drugs of abuse have been reported. However, few have evaluated stability in frozen urine samples stored for 12 months. We have determined the stability of 11-nor-9-carboxy-delta 9-tetrahydrocannabinol (9-COOH-THC), amphetamine, methamphetamine, morphine, codeine, cocaine, benzoylecgonine, and phencyclidine in 236 physiological urine samples. Following the initial quantitative analysis, the samples were stored at -20 degrees C for 12 months and then reanalyzed. All drug concentrations were determined by gas chromatographic-mass spectrometric methods with cutoff concentrations of 5 ng/mL for 9-COOH-THC and phencyclidine and 100 ng/mL for each of the other drugs. The average change in the concentrations of these drugs following this long-term storage was not extensive except for an average change of -37% in cocaine concentrations.

Factors affecting the loss of carbon monoxide from stored blood samples.

The loss of carbon monoxide (CO) from whole blood or hemolysates has been investigated. Blood samples were exposed to the atmosphere or to a limited volume of air for various storage periods at three temperatures. The initial hemoglobin (Hb) concentration and the percent carboxyhemoglobin (%COHb) saturation were varied in separate experiments. In addition, the effect of repeated exposure of blood to air was evaluated. The %COHb saturation decreased from 80 to 50% following storage of a 1-mL blood sample with 49 mL of air in a sealed container at room temperature for 45 hr. Greater decreases in the %COHb saturation were observed in samples which were exposed to the atmosphere. Lesser, but significant, losses occurred when samples were stored in a refrigerator or freezer. The concentration of Hb in the samples as well as the initial %COHb saturation were found to influence the decrease in the %COHb saturation.

Determination of carbon monoxide in blood by gas chromatography using a thermal conductivity detector.

A method is described for the gas chromatographic quantitation of carbon monoxide by means of thermal conductivity detection. Carbon monoxide is released from blood samples as small as 0.02 mL using a unique extraction chamber. The method was compared to a standard gas chromatographic and spectrophotometric method of carbon monoxide quantitation. It was comparable to the former with all samples evaluated and apparently more reliable than the latter with decomposed samples.

Detection of opiates in postmortem tissues by thin-layer immunoassay.

The use of thin-layer immunoassay (TIA) for the detection of opiates in postmortem brain, kidney, liver, and lung tissue has been developed. The method is simple to perform, requires no specialized equipment or radioactive reagents, and is sufficiently sensitive for the detection of opiates in postmortem tissues. Sample preparation consists of homogenization followed by ultradialysis to remove protein. The procedure was evaluated in a blind study of 83 postmortem tissue specimens and found to be reliable at tissue concentrations greater than or equal to 200 ng/g as determined by radioimmunoassay (RIA).

The detection of protein p30 in seminal stains by means of thin-layer immunoassay.

The detection of p30 by means of an indirect thin-layer immunoassay (TIA) is described. Extracts from 20 samples can be analyzed in approximately 2 h with a detection limit of approximately 50 ng. The p30 protein was detected in seminal stains which had been stored at room temperature for six months and at 130 degrees C for 4 h. Blood, saliva, urine, perspiration, and tears did not interfere with the method. The reliability of the method was demonstrated in a blind study.

The detection of fetal hemoglobin in bloodstains by means of thin-layer immunoassay.

A method for the detection of fetal hemoglobin in bloodstains by means of thin-layer immunoassay is described. The equivalent of 0.01 microL of blood containing 0.18 to 0.24 microgram of fetal hemoglobin may be detected by this method. Studies with stains up to two years old and blind studies have shown these methods to be sufficiently sensitive and specific to be of value in forensic serology.

Detection of opiates in blood by thin-layer immunoassay.

The use of thin-layer immunoassay (TIA) for the detection of opiates in postmortem blood samples has been developed. Samples required no preparation other than protein removal, which was accomplished easily by the use of a commercially available ultrafiltration device. The procedure was evaluated in a blind study of 82 postmortem blood samples and found to be reliable. Opiate was detected in these samples at a RIA-determined concentration as low as 10 ng/mL.

Detection of benzoylecgonine in urine by means of UV spectrophotometry.

A simple and reliable method has been developed for the detection of benzoylecgonine in urine by UV spectrophotometry. Benzoylecgonine was isolated by liquid-liquid extraction and subsequently identified based on its hydrolysis by NaOH. The decrease in absorbance at 235 nm caused by this NaOH hydrolysis was used to identify benzoylecgonine even in the absence of its characteristic spectrum. None of the other drugs evaluated interfered with the method, which has a detection limit of 1.25 micrograms/mL.

Detection of opiates in urine by means of thin-layer immunoassay.

The use of thin-layer immunoassay (TIA) for the direct detection of opiates in unextracted urine is described. This novel immunoassay is capable of detecting less than 1 ng of opiates, rapidly and inexpensively, without the use of radioactive materials or sophisticated analytical instrumentation. The method was evaluated in a study of 68 post mortem urines and found to be reliable for the detection of opiates in this type of sample.

The identification of human bloodstains by means of a micro-thin-layer immunoassay procedure.

A method for the identification of human bloodstains using a micro-thin-layer immunoassay (TIA) procedure is presented. The equivalent of approximately 0.01 micro /leter of human blood may be detected using this method with anti-human hemoglobin. Blind studies and stability studies indicate that the method is sufficiently specific and sensitive to be of forensic science value.