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R J Letcher

Publications and source records attributed to R J Letcher.

11 recordsLinked to original sources
PubMed2001

Metabolism of PCBs by the deepwater sculpin (Myoxocephalus thompsoni).

Methylsulfonyl-PCBs (MeSO2-PCBs) are hydrophobic organic contaminants that bioaccumulate in the environment similar to their parent molecules, PCBs. Previously, MeSO2-PCBs have primarily been identified in tissues of birds, humans, and other mammals. However, evidence now supports formation of these metabolites in deepwater sculpin, Myoxocephalus thompsoni, a benthic forage fish predominant in the Great Lakes. The ability of deepwater sculpin to form MeSO2-PCBs is unprecedented for a freshwater fish species and presents a novel biochemical pathway for organochlorine metabolism. Additionally, this appears to be a unique PCB metabolic pathway resulting in a reduction of as much as 10% in the sculpin PCB burden, which is further transformed into another class of organic contaminants in the Great Lakes ecosystem.

Animals↗
PubMed2001

Indications for the involvement of a CYP3A-like iso-enzyme in the metabolism of chlorobornane (Toxaphene) congeners in seals from inhibition studies with liver microsomes.

The different isoforms of the cytochrome P450 (CYP) system can metabolise a suite of classes of lipophilic, anthropogenic compounds. The bioaccumulative potential as well as the toxicity of xenobiotics may be significantly altered in the process. To compare the metabolic ability of different wildlife species, it is important to identify the different iso-enzymes of CYP, which are responsible for the metabolism of different classes of compounds. This can be achieved with in vitro incubation assays. In the present study, preparations of hepatic microsomes of a harbour seal (Phoca vitulina) and a grey seal (Halichoerus grypus) demonstrated that the chlorobornane (CHB) congeners CHB-32 and -62 were metabolised enzymatically to their hydroxylated derivatives. These derivatives were partially characterised by their NCI mass-spectra. Inhibition studies were carried out to identify the specific CYP isoform(s) responsible for the metabolism of CHB-32 and -62. Ketoconazole has been shown to inhibit CYP3A enzymes in human and rat studies. In this study, ketoconazole caused concentration-dependent inhibition of metabolism of CHB-32 and -62, reaching 80% at the 1.0 microM treatment level. Ellipticine (1.0 microM), which has been shown to inhibit CYP1A1/2, also inhibited CHB-32 and -62 metabolism in the microsomes of grey seal, but to a much lower degree of less than 10 and 24%, respectively. In the same experiment the metabolism of 4,4'-dichlorobiphenyl was already inhibited 70% by ellipticine treatment at the same concentration. This non-ortho substituted PCB congener can easily attain a planar molecular configuration, and therefore served as a model CYP1A substrate. Inhibition of chlorobornane metabolism was not observed after the addition of goat anti-rat CYP2B antibodies or Aldrin, which is a model CYP2B substrate in rat. Cautious interpretation is advised for results obtained with so-called selective competitive inhibitors. Regardless, these studies indicated for the first time the possible involvement a CYP3A isoform in the mediation of chlorobornane metabolism in seals. The immunochemical cross-reactivity of mouse, rabbit or sheep anti-rat antibodies in the hepatic microsomes of harbour seal confirmed the presence of CYP1A1/2, CYP1A1, CYP2B1/2, CYP3A and CYP4A isoenzymes. Enantioselective metabolism by the microsomes of harbour seal was observed for both CHB-32 and -62. Stereochemical preferences of biotransformation enzymes can have an influence on the environmental distribution of both enantiomers of optically active compounds.

Aldrin↗
PubMed2001

Effects of chloro-s-triazine herbicides and metabolites on aromatase activity in various human cell lines and on vitellogenin production in male carp hepatocytes.

We investigated a potential mechanism for the estrogenic properties of three chloro-s-triazine herbicides and six metabolites in vitro in several cell systems. We determined effects on human aromatase (CYP19), the enzyme that converts androgens to estrogens, in H295R (adrenocortical carcinoma), JEG-3 (placental choriocarcinoma), and MCF-7 (breast cancer) cells; we determined effects on estrogen receptor-mediated induction of vitellogenin in primary hepatocyte cultures of adult male carp (Cyprinus carpio). In addition to atrazine, simazine, and propazine, two metabolites--atrazine-desethyl and atrazine-desisopropyl--induced aromatase activity in H295R cells concentration-dependently (0.3-30 microM) and with potencies similar to those of the parent triazines. After a 24-hr exposure to 30 microM of the triazines, an apparent maximum induction of about 2- to 2.5-fold was achieved. The induction responses were confirmed by similar increases in CYP19 mRNA levels, determined by reverse-transcriptase polymerase chain reaction. In JEG-3 cells, where basal aromatase expression is about 15-fold greater than in H295R cells, the induction responses were similar but less pronounced; aromatase expression in MCF-7 cells was neither detectable nor inducible under our culture conditions. The fully dealkylated metabolite atrazine-desethyl-desisopropyl and the three hydroxylated metabolites (2-OH-atrazine-desethyl, -desisopropyl, and -desethyl-desisopropyl) did not induce aromatase activity. None of the triazine herbicides nor their metabolites induced vitellogenin production in male carp hepatocytes; nor did they antagonize the induction of vitellogenin by 100 nM (EC(50) 17beta-estradiol. These findings together with other reports indicate that the estrogenic effects associated with the triazine herbicides in vivo are not estrogen receptor-mediated, but may be explained partly by their ability to induce aromatase in vitro.

Animals↗
PubMed2001

In vitro estrogenicity of polybrominated diphenyl ethers, hydroxylated PDBEs, and polybrominated bisphenol A compounds.

Polybrominated diphenyl ethers (PBDEs) are used in large quantities as additive flame retardants in plastics and textile materials. PBDEs are persistent compounds and have been detected in wildlife and in human adipose tissue and plasma samples. In this study, we investigated the (anti)estrogenic potencies of several PBDE congeners, three hydroxylated PBDEs (HO-PBDEs), and differently brominated bisphenol A compounds in three different cell line assays based on estrogen receptor (ER)-dependent luciferase reporter gene expression. In human T47D breast cancer cells stably transfected with an estrogen-responsive luciferase reporter gene construct (pEREtata-Luc), 11 PBDEs showed estrogenic potencies, with concentrations leading to 50% induction (EC(50)) varying from 2.5 to 7.3 microM. The luciferase induction of the most potent HO-PBDE [2-bromo-4-(2,4,6-tribromophenoxy)phenol] exceeded that of estradiol (E(2)), though at concentrations 50,000 times higher. As expected, brominated bisphenol A compounds with the lowest degree of bromination showed highest estrogenic potencies (EC(50) values of 0.5 microM for 3-monobromobisphenol A). In an ER alpha-specific, stably transfected human embryonic kidney cell line (293-ER alpha-Luc), the HO-PBDE 4-(2,4,6-tribromophenoxy)phenol was a highly potent estrogen with an EC(50) < 0.1 microM and a maximum 35- to 40-fold induction, which was similar to E(2). In an analogous ER beta-specific 293-ER betas-Luc cell line, the agonistic potency of the 4-(2,4,6-tribromophenoxy)phenol was much lower (maximum 50% induction compared to E(2)), but EC(50) values were comparable. These results indicate that several pure PBDE congeners, but especially HO-PBDEs and brominated bisphenol A-analogs, are agonists of both ER alpha and ER beta receptors, thus stimulating ER-mediated luciferase induction in vitro. These data also suggest that in vivo metabolism of PBDEs may produce more potent pseudoestrogens.

Air Pollutants, Occupational↗
PubMed1999

Cytotoxicity and aromatase (CYP19) activity modulation by organochlorines in human placental JEG-3 and JAR choriocarcinoma cells.

The human placental JEG-3 and JAR choriocarcinoma cell lines have been used as placental models for the study of aromatase (CYP19) activity and endocrine functions. In the present study, 21 organochlorines (OCs) mediated decreases in aromatase activity and protein and DNA content and increases in the percent lactate dehydrogenase (LDH) leakage in JEG-3 cells. These effects were highly variable among the types of OC and their treatment concentrations. Lowest observed effective concentrations reached 0. 001 microM for several OCs. Aromatase activity decreases and OC-mediated cytotoxicity were related. Thus, it was not possible to clearly assess the capacity of the OCs to modulate aromatase activity. Similar to 1,4-naphthoquinone, the most cytotoxic OCs contained a hydroxyl (4'-OH-2,4,6-trichlorobiphenyl and tris(4-chlorophenyl)methanol) or methylsulfonyl- (3- and 4-MeSO(2)-2, 2',5,5'-tetrachlorobiphenyl and -2,3',4',5-tetrachlorobiphenyl, and 3'- and 4'-MeSO(2)-2,2',3,4,5'-pentachlorobiphenyl and -2,2',4,5, 5'-pentachlorobiphenyl) functional group. Modulation of aromatase activity and LDH leakage were less for 3,3',4,4', 5-pentachlorobiphenyl and benzo[a]pyrene and insignificant for five alkyl-substituted trichloro-dibenzofurans and 2,3,7, 8-tetrachloro-dibenzo-p-dioxin (up to 10 microM). Cytotoxicity-related effects were influenced by the cell density and the presence of 10% fetal calf serum in the medium during compound incubation. Similar cytotoxic effects were observed for the JAR cell line. The involvement of an apoptotic mechanism of cytotoxicity in OC-treated JEG-3 cells was suggested by the binding of APO2.7 (an antibody specific to apoptotic cells), DNA fragmentation, and trypan blue staining. JEG-3 and JAR cells appear too sensitive toward OC-mediated cytotoxicity for use as in vitro bioassays to evaluate the potential modulation of aromatase activity. However, these cell lines may prove useful for examining the capacity of xenobiotics to modulate placental toxicity.

Apoptosis↗
PubMed1996

Immunoquantitation and microsomal monooxygenase activities of hepatic cytochromes P4501A and P4502B and chlorinated hydrocarbon contaminant levels in polar bear (Ursus maritimus).

Contamination of the Arctic ecosystem by anthropogenic compounds has resulted in exposure of polar bear (Ursus maritimus) to lipophilic chlorinated hydrocarbon contaminants (CHCs) accumulated through the marine food web. Liver samples were collected from 16 adult male polar bears in the Canadian arctic and subjected to chemical analysis for CHCs and metabolites, determination of alkoxyresorufin O-dealkylase activities, and immunoquantitation of cytochrome P450 (CYP) protein levels. We report on the relationships between the hepatic microsomal levels of immunoreactive CYP1A and CYP2B isozymes, catalytic activities, and hepatic CHC and metabolite concentrations in polar bear. We specifically explored the influence of several CHCs on the induction of hepatic CYP in polar bear and the potential use of immunoassay quantitation as a bioindicator of CHC exposure. Polychlorinated biphenyls (PCB) classed as CYP1A and mixed CYP1A/CYP2B inducers accounted for about 25% of the total PCB residues present (18,680 +/- 5053 ng/g lipid). CYP1A protein content correlated strongly with hepatic levels of PCBs, PCDDs (0.032 +/- 0.018 ng/g lipid, and PCDFs (0.011 +/- 0.007 ng/g lipid) and their corresponding toxic equivalents (TEQ, 0.377 +/- 0.182 ng/g lipid). Mono-ortho-CB-156, CB-157, and CB-105 were the predominant TEQ contributors. Correlations between CYP2B protein content and CHC residue levels in polar bear liver suggested that ortho-chlorine-substituted PCBs and chlordanes were the major contributors to CYP2B induction. CYP1A and CYP2B contents were therefore good indicators of CHC exposure in polar bear liver. Ethoxyresorufin, pentoxyresorufin, and benzyloxyresorufin O-dealkylase activities increased with increasing CYP1A protein content up to protein levels of approximately 5 pmol/mg, suggesting that all three activities were primarily CYP1A-mediated. These results were substantiated by antibody inhibition experiments. In summary, immunoquantitated CYP1A and CYP2B isozymes are a more reliable measure of exposure to CHC inducers than alkoxyresorufin O-dealkylase activities in polar bear.

Animals↗
PubMed1995

An integrated analytical method for determination of polychlorinated aryl methyl sulfone metabolites and polychlorinated hydrocarbon contaminants in biological matrices.

A simple column chromatography method was developed for separation and cleanup in the determination of chlorinated hydrocarbon contaminants and their methyl sulfone (MeSO2-) metabolites in biological tissues. The method was validated for determination of 11 polychlorinated biphenyls (PCBs), 15 tetra- to heptachloro 3- and 4-MeSO2-PCBs, 3-MeSO2-DDE, and tris(4-chlorophenyl)-methanol spiked to herring gull egg, smelt, and polar bear liver and adipose tissue using gas chromatography with electron-capture detection (GC-ECD). The overall mean recovery relative to the internal standard was 103% +/- 8%, independent of analyte, substrate type, and lipid extract weights up to approximately 0.7 g. Precision of replicate analyses of individual congeners was good. There were no significant residual biogenic or xenobiotic interferences in the aryl methyl sulfone fraction of any substrate. Sensitivity and linearity of molar response of MeSO2-PCBs and MeSO2-DDE was tested for ECD and electron-capture negative ion mass spectrometry monitoring the total ion current (TIC) and the molecular ion (SIM). The mean practical quantitation limit among MeSO2-PCBs and 3-MeSO2-DDE was lowest for SIM (2.1 +/- 0.9 pg) and similar for ECD and TIC (24.2 +/- 4.6 and 44.4 +/- 17.1 pg, respectively). Response factors were linear above the practical quantitation limit to at least the nanogram level for all three techniques. In spite of superior sensitivity, there was more inherent variability in the response factors for SIM (approximately 18%-56% CV) than for ECD (approximately 7%-12% CV) or TIC (approximately 11%-18% CV); therefore, ECD or TIC is recommended for quantitative analysis.

Animals↗
PubMed1995

Geographical distribution and identification of methyl sulphone PCB and DDE metabolites in pooled polar bear (Ursus maritimus) adipose tissue from western hemisphere arctic and subarctic regions.

The geographical distribution of methyl sulphone (MeSO2-) PCB and DDE metabolites is unknown for any species in any region. In this study, pooled male polar bear (Ursus maritimus) adipose samples from 12 arctic and subarctic regions from the Bering Sea to the Greenland Sea were analyzed for PCBs, DDE, and their MeSO2-metabolites. Each adipose pool contained from three to 27 lipid extracts prepared on an equal fat basis. The total (S-) MeSO2-PCB and MeSO2-DDE levels were generally higher in the east (Greenland) and south (Hudson Bay) and ranged from 138 to 633 ng/g lipid and 0.8-11 ng/g lipid, respectively. Five new MeSO2- compounds were identified and/or quantitated, 3M-DDE, 4M-CB64, 3M- and 4M-CB70, and 3M-CB149. The congeners 3M- and 4M-CB101 and 3M- and 4M-CB87 consistently accounted for ca. 50% of the S-MeSO2-PCBs. The MeSO2-PBC congener pattern was independent of geographic variation among the regions, implying that the pattern of atmospheric input and subsequent biomagnification of precursor PCBs is homogeneous in the western hemisphere. The ratio of S-MeSO2-PCBs to accumulating PCBs decreased very gradually (average value of .056 +/- .012) from west to east reflecting the proportion of higher chlorinated PCBs that are not major precursors of MeSO2-PCBs. The 3-MeSO2-DDE/DDE ratio fluctuated among regions with an average value of .033 +/- 0.017 [corrected].

Adipose Tissue↗
PubMed1995

Preliminary results of fasting on the kinetics of organochlorines in polar bears (Ursus maritimus).

We determined concentrations of polychlorinated biphenyls (PCBs), chlordanes (CHLORs), chlorobenzenes (CBzs), hexachlorocyclohexanes (HCHs), and dichlorodiphenyltrichloroethane (DDT) and its metabolites (DDD and DDE) in the tissues of individual polar bears (Ursus maritimus) before and after a lengthy period of fasting. Polar bears are an ideal model for such studies. They undergo one of the most extreme fasts known for any mammal and are located at the top of a long food chain, thus biomagnification of organochlorines (OC) is significant. Adipose tissue and milk were collected from different reproductive classes of adult females (solitary/pregnant, with cubs-of-the-year, with yearling cubs) and were analyzed for organochlorine content. As the fasting period progressed and adipose reserves decreased, concentrations of some organochlorines in the adipose tissue and milk increased on a lipid weight basis. The transfer of contaminants from mothers to offspring thus also increases with duration of the fasting period. This phenomenon could adversely influence the survival and growth of cubs during the critical early phase of their development.

Adipose Tissue↗
PubMedDate not supplied

Immunoquantitation of cytochromes P450 1A and P450 2B and comparison with chlorinated hydrocarbon levels in archived polar bear liver samples.

The present study examined the utility of an immunoblot method for quantitation of cytochrome P450 isozymes in archived liver samples as a bioassay of exposure to halogenated hydrocarbons. Hepatic microsomes were prepared from 44 archived polar bear (Ursus maritimus) liver homogenates that had been stored at approximately -40 degrees C for 9-10 years and analyzed on blots probed with antibodies to rat cytochromes P450 1A1 and P450 2B1. The results revealed a positive correlation between cytochrome P450 1A and total polychlorinated biphenyl (PCB) levels in the archived liver samples, suggesting that cytochrome P450 1A was induced in polar bears by environmental exposure to PCBs.

Animals↗