Characterization of mass-labeled [13C14]-decabromodiphenylethane and its use as a surrogate standard in the analysis of sewage sludge samples.

Very little data is available about the presence of the brominated flame retardant, DBDPE, in the environment. This study reports the characterization of [(13)C(14)]-decabromodiphenylethane and the use of this surrogate standard to positively identify and quantify the presence of DBDPE in sewage sludge samples. The large difference in response factors between BDE-209 and DBDPE predicates the use of [(13)C(14)]-decabromodiphenylethane as a surrogate standard to improve the accuracy when determining the levels of DBDPE in environmental samples.

Modulatory effects of neonatal exposure to TCDD, or a mixture of PCBs, p,p'-DDT, and p-p'-DDE, on methylnitrosourea-induced mammary tumor development in the rat.

The role of organochlorine (OC) exposure in the etiology of breast cancer remains controversial. Thus, our objective was to determine whether the most abundant and toxic OCs found in human milk could, when ingested during the neonatal period, modulate the development of mammary tumors in the rat. We prepared a mixture composed of p,p'-dichlorodiphenyltrichloroethane (DDT), its major metabolite, p,p'-dichlorodiphenyldichloroethene (DDE), and 19 polychlorinated biphenyls (PCB) based on their concentrations found in the milk of Canadian women. Neonate rats at 1, 5, 10, 15, and 20 days of age were gavaged with this mixture, at 10, 100, and 1,000 times the amount that a human baby would consume. An additional group received 2.5 microg 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)/kg body weight (bw) by gavage at 18 days of age, instead of the mixture. On day 21, all treatment groups, except for a control group and a 1,000-mix group, received a single intraperitoneal injection of methylnitrosourea (MNU, 30 mg/kg bw), the initiator of the carcinogenic process. The average number of rats per treatment group was 33. Rats were sacrificed when their tumors reached 1 cm in size, or at 308 days of age. We prepared mammary tumors and mammary gland whole mounts for histologic analysis. There were no significant effects when only the malignant or only the benign tumors were considered. After all benign and malignant lesions were pooled, the number of mammary tumors differed among all MNU-treated groups (p = 0.02) with more lesions developing in the MNU-1,000[times] (median = 4.5; p = 0.05) and MNU-TCDD (median = 5.5; p = 0.07) compared to the MNU-0 rats (median = 2). Compared to the MNU-0 group, the percentage of rats that developed palpable tumors (benign plus malignant) was slightly higher (p = 0.06) in the MNU-TCDD group, but not in the MNU-1,000[times] group. The percentage of palpable tumors that were malignant was higher (p = 0.02) in the MNU-100[times] group (15/16, 94%) than in the MNU-0 group (10/18, 56%). The highest dose of the mixture delayed (p = 0.03) the development of tumors, but this was not observed with the MNU-TCDD treatment. These results suggest that neonatal exposure to high doses of organochlorines could favor the development of MNU-induced mammary lesions, but also delays the development of palpable tumors in the rat.

Effects of gestational and lactational exposure to Aroclor 1242 on sperm quality and in vitro fertility in early adult and middle-aged mice.

The objective of this study was to examine the effects of gestational and lactational exposure to Aroclor 1242 (0, 10, 25, 50, and 100 mg/kg-bw) on male fertility. Doses were administered to C57BL6 female mice orally every two days from two weeks before mating, during mating, and through gestation until postnatal day 21. Male B6D2F1 offspring were examined for anogenital distance, organ development, epididymal sperm count, sperm motility, and in vitro fertility at 16 and 45 weeks of age. Stomach samples of pups nursing from PCB-treated mothers in the 50 mg/kg dose group were analyzed for PCBs and chlorobiphenylols by high resolution gas chromatography coupled with low resolution mass spectrometry. It was estimated that the nursing pups were exposed to 0.2, 0.6, 1.2, and 2.4 mg/kg/day total PCBs in the 10, 25, 50, and 100 mg/kg dose groups, respectively. This exposure level approaches the maximum FDA recommended levels for PCBs in food and breast milk. The composition of the PCBs in the stomach samples was different from the parent mixture, as there was a higher proportion of heavily chlorinated congeners, as well as chlorobiphenylols. Anogenital distance at weaning, and liver, thymus, and testes weight at 16 and 45 weeks of age were not affected by PCB exposure. Epididymal sperm velocity and linearity were significantly increased in the 25 mg/kg dose group at 16 weeks of age. Sperm count was increased by 36% in this dose group (P = 0.06). By 45 weeks of age, average sperm count in this dose group was similar to that of controls. With the exception of the 50 mg/kg dose group at 16 weeks of age, sperm fertilizing ability in vitro was significantly decreased in all PCB-exposed groups at 16 and 45 weeks of age. These results suggest that fertility in the adult mouse is susceptible to developmental exposure to Aroclor 1242 and is independent of testis weight or epididymal sperm count.

Synthesis of polybrominated diphenyl ethers and their capacity to induce CYP1A by the Ah receptor mediated pathway.

Polybrominated diphenyl ethers (PBDEs) have become widely distributed as environmental contaminants due to their use as flame retardants. Their structural similarity to other halogenated aromatic pollutants has led to speculation that they might share toxicological properties such as hepatic enzyme induction. In this work we synthesized a number of PBDE congeners, studied their affinity for rat hepatic Ah receptor through competitive binding assays, and determined their ability to induce hepatic cytochrome P-450 enzymes by means of EROD (ethoxyresorufin-O-deethylase) assays in human, rat, chick, and rainbow trout cells. Both pure PBDE congeners and commercial PBDE mixtures had Ah receptor binding affinities 10(-2)-10(-5) times that of 2,3,7,8-tetrachlorodibenzo-p-dioxin. In contrast with polychlorinated biphenyls, Ah receptor binding affinities of PBDEs could not be related to the planarity of the molecule, possibly because the large size of the bromine atoms expands the Ah receptor's binding site. EROD activities of the PBDE congeners followed a similar rank order in all cells. Some congeners, notably PBDE 85, did not follow the usual trend in which strength of Ah receptor binding affinity paralleled P-450 induction potency. Use of the gel retardation assay with a synthetic oligonucleotide indicated that in these cases the liganded Ah receptor failed to bind to the DNA recognition sequence.

Examination of the estrogenicity of 2,4,6,2',6'-pentachlorobiphenyl (PCB 104), its hydroxylated metabolite 2,4,6,2',6'-pentachloro-4-biphenylol (HO-PCB 104), and a further chlorinated derivative, 2,4,6,2',4',6'-hexachlorobiphenyl (PCB 155).

Several studies have reported that polychlorinated biphenyls (PCBs) exhibit estrogenic activity; however, it is not clear if these responses are associated with the polychlorinated hydrocarbon or its hydroxylated metabolite. In order to further test this hypothesis, a battery of in vitro and in vivo assays were used to investigate the estrogenic and antiestrogenic activities of 2,4,6,2',6'-pentachlorobiphenyl (PCB 104), its para-hydroxylated derivative 2,4,6,2',6'-pentachloro-4-biphenylol (HO-PCB 104), and its para-chlorinated derivative 2,4,6,2',4',6'-hexachlorobiphenyl (PCB 155). PCB 104 was found to 1) compete with tritiated 17beta-estradiol (E2) for binding to the mouse uterine estrogen receptor (ER); 2) induce gene expression in MCF-7 human breast cancer cells transiently transfected with the Gal4-human ER chimeric construct (Gal4-HEGO) and the Gal4-regulated luciferase reporter gene (17m5-G-Luc); and 3) increase MCF-7 cell proliferation in a dose-dependent manner. HO-PCB 104 exhibited greater estrogenic activity than PCB 104 in the in vitro assays examined. However, gas chromatographic-mass spectrophotometric analysis of extracts prepared from MCF-7 cells incubated with PCB 104 failed to detect the presence of the expected major metabolite HO-PCB 104. The estrogenic activity of the para-chlorinated derivative, PCB 155, was minimal compared to PCB 104 and HO-PCB 104, but it did exhibit significant antiestrogenic activity following co-treatment with 1 nM E2. Co-treatment of PCB 104 with 1 nM E2 had no effect on reporter gene expression compared to E2 alone, while 10 microM HO-PCB 104 exhibited additivity with 1 nM E2. At a dose of 202 mg/kg,PCB 104 increased uterine wet weight in ovariectomized CD-1 mice and induced vaginal epithelial cell cornification at 202, 16, and 1.7 mg/kg in a dose-dependent manner. These studies demonstrate that in addition to the hydroxylated metabolites, selected parent PCB congeners may also exhibit estrogenic and antiestrogenic activities.

Effects of individual terphenyls and polychlorinated terphenyls on rat hepatic microsomal cytochrome P-450-dependent monooxygenases: structure-activity relationships.

The effects of o-, m- and p-terphenyl, 2,4-dichloro-, 2,4,6-trichloro-, 2,3,5,6-tetrachloro-, 2,3,4,6-tetrachloro-, 2,4,4''',6- tetrachloro- and 2,3,4,5-tetrachloro-p-terphenyl, 2,3,4,5-tetrachloro-m- and o-terphenyl as inducers of hepatic drug-metabolizing enzymes were determined in immature male Wistar rats. o-Terphenyl, 2,4-dichloro-, 2,4,6-trichloro-p-terphenyl and 2,3,4,5-tetrachloro-o-terphenyl induced 4,4'-dimethylamino antipyrine N-demethylase at total dose levels of 300 mumol/kg and the 2,3,4,5-tetrachloro-p-terphenyl induced ethoxyresorufin O-deethylase (EROD). In contrast, none of the other terphenyls or polychlorinated terphenyls (PCTs) induced these enzyme activities. Previous studies have demonstrated that 2,3,4,5-tetrachloro-p-terphenyl did not exhibit a high affinity for the 2,3,7,8-tetrachlorodibenzo-p-trachlorodibenzo-p-dioxin (TCDD) receptor protein (EC50 = 6.6 X 10(-6) M). In contrast, this study showed that 2,3,4,5-tetrachloro-p-terphenyl was more active than either 2,3,4,5-tetrachloro-o- or m-terphenyl as an inducer of EROD. Moreover, the competitive receptor binding EC50 values for the latter two isomers were greater than 10(-5) M and this result was also consistent with their lack of EROD induction activity. Previous studies showed that analysis of the data for a series of 4'-substituted-2,3,4,5-tetrachlorobiphenyls indicated that the p-terphenyl structural moiety (i.e. 4'-substituent = phenyl) did not interact with high affinity with the receptor protein binding site. Since the 2,3,4,5-tetrachloro o- and m-terphenyls are also poor ligands for the receptor protein, this data and results from other studies indicate that PCT congeners (and commercial mixtures) are therefore unlikely to elicit significant 2,3,7,8-TCDD-like biologic or toxic effects in target species.

Gas chromatographic/mass spectrometric analysis of specific isomers of polychlorodibenzofurans.

The gas chromatographic/mass spectrometric characteristics of 26 congeners of polychlorinated dibenzofurans, previously characterized by specific synthetic routes and by standard spectroscopic techniques, have been evaluated. The electron impact mass spectra are not particularly isomer-specific, though 2,3,7,8-tetrachlorodibenzofuran is distinguishable on this basis from the three other tetrachloro isomers investigated in this work. Positive ion methane chemical ionization mass spectra do show a greater degree of isomer distinction, and are reasonably reproducible. Electron attachment negative ion spectral characteristics are also presented. Preliminary results on negative ion chemical ionization mass spectra, obtained using methane plus small amounts of oxygen as reagent gas, are reported.

Formation of polychlorinated dibenzofurans and dioxins during combustion, electrical equipment fires and PCB incineration.

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) are likely formed by thermal synthesis of a variety of primary precursors. Highest levels of these compounds are expected, however, when the starting material requires only one or two reaction steps for their formation, as is the case with chlorophenols, chlorobenzenes and polychlorinated biphenyls (PCBs). Laboratory pyrolyses have indeed shown that PCBs give significant yields of PCDFs, and chlorobenzenes give both PCDFs and PCDDs. In addition, a variety of other chloroaromatic compounds are formed. From these experiments and from accidents involving PCB fires, it is known that PCDFs are the most important toxic compounds associated with PCBs. Most commercial PCBs contain PCDFs in the low ppm range. PCDF concentration does not increase during normal operation in electrical equipment. Accidents (fires and explosions) involving PCBs can give PCDF levels in soot of up to 1000 ppm and higher. Effective thermal destruction of PCB is possible in modern incineration units, provided high temperatures, excess air and sufficient residence times are used. Exact figures for minimum temperature and residence time cannot be given, since feedstock and incinerator construction greatly influence destruction efficiency. Effluents from EPA-licensed incinerators used for PCB destruction contain only very low levels of PCDDs and PCDFs.

Aryl hydrocarbon hydroxylase (AHH) induction by polybrominated biphenyls (PBBs): enchancement by photolysis.

Irradiation of the commercial polybrominated biphenyl (PBB( mixture, fireMaster BP-6, in cyclohexane solution at 300 nm for 930 min resulted in a marked diminution of the major components of the mixture. Administration of the photolyzed PBB mixture of fireMaster BP-6 to immature male Wistar rats caused both dose-related decreases in thymus weight and increase in hepatic microsomal benzo[a]pyrene hydroxylase (AHH), 4-dimethylaminoantipyrine N-demethylase and NADPH-cytochrome c reductase activities and cytochrome P-450 content. The dose effecting half-maximal AHH induction for the photolyzed PBBs (9 mg . KG-1) was approximately 6 times lower than that of fireMaster BP-6 (50 mg. kg-1). Furthermore, the concentration of photolyzed PBBs (2 micrometers) required to displace 50% of the specifically-bound [3H] TCDD from its high-affinity cytosolic Ah receptor was approximately 150 times lower than that required for fireMaster BP-6 (300 micrometers), as measured by sucrose density gradient centrifugation analysis. The results suggest that the photolysis of the commercial PBB mixture yields products which possess increased biologic activity.