Interactions between 2,3,7,8-TCDD and PCBs as tumor promoters: limitations of TEFs

The assessment of the carcinogenic risk of polychlorinated dioxins (PCDDs), furans (PCDFs), and biphenyls (PCBs) by TEFs is hampered by species- and tissue-specific responses that cannot readily be explained by differences in the Ah receptor levels but may be due to events subsequent to ligand binding to the Ah receptor. Moreover, PCDDs and related compounds accumulate in the environment, in animal and human tissues as highly complex mixtures. Thus, comprehensive risk assessment should include all Ah receptor ligands and agents that modulate the Ah receptor-mediated responses. Tumor promoter studies with mixtures of PCDDs and halogenated biphenyls have shown additive, synergistic, and antagonistic effects. To analyse the interactions of TCDD and PCBs as tumor promoters in more detail, we established an in vitro assay, i.e., the enhancement (promotion) of malignant transformation of carcinogen-initiated C3H/M2 mouse fibroblasts after treatment with tumor promoters. The coplanar PCB 126, a potent Ah receptor agonist, and the diortho-substituted PCB153, to which no TEF value has been ascribed, are promoters of malignant transformation. A defined mixture of PCB126 and TCDD had an additive promoting effect, while PCB 153 antagonized the TCDD-mediated promotion. Thus, the TEF-approach may be insufficient to estimate the tumor-promoting activities of PCDDs, PCDFs, and PCBs in mammalian tissues in which diortho-substituted PCBs are greatly accumulated.     

Ethoxyresorufin-O-deethylase (EROD) inducing potencies of planar chlorinated aromatic hydrocarbons in primary cultures of hepatocytes from different developmental stages of the chicken

In vitro induction of ethoxyresorufin O-deethylase (EROD) activity in cell cultures is an extensively validated tool for measuring overall potencies of mixtures of halogenated aromatic hydrocarbons (HAHs) in samples from the abiotic or biotic environment. For risk assessment with special attention to effects in wild birds, an assay was developed that makes use of chicken embryo hepatocytes. However, it was questioned whether compound-specific responses are consistent at the various developmental stages. The results of our present study show that there are considerable differences between early and late embryonal and post-hatching stages. The induction of EROD was measured in primary chicken hepatocyte cultures. The cells were isolated at day 14 and day 19 of embryonal development and at day 1 post hatching. Hepatocytes were exposed in vitro to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB 126, IUPAC nomenclature) and 2,3',4,4',5-pentachlorobiphenyl (PCB 118). The respective compounds were chosen as representatives for dioxins, furans, non-ortho PCBs, and mono-ortho PCBs. These groups of chemicals have been identified as environmental contaminants with major dioxin-like effects that are mediated by a common receptor, the arylhydrocarbon (Ah) receptor. At all developmental stages, TCDF was more potent than TCDD. Relative potencies (RP = EC50TCDD/EC50HAH) decreased in the order TCDF < TCDD < PCB 126 < PCB 118. Depending on the developmental stage, TCDF was 1.2 to 3.4 times more potent than TCDD. PCB 126 was equipotent or less potent by a factor of 3 than TCDD. PCB 118 was 100 to 300 times less potent than TCDD. Both the mean effective concentration (EC50) and the maximum EROD activity (Ymax) of all compounds were lower in hepatocyte cultures from 14-day-old embryos than those from 19-day-old embryos or 1-day-old hatchlings. RPs were comparable in 19-day-old embryos and in hatchlings, but significantly different in 14-day-old embryos.     

Profiles of Great Lakes critical pollutants: a sentinel analysis of human blood and urine. The Great Lakes Consortium

To determine the contaminants that should be studied further in the subsequent population-based study, a profile of Great Lakes (GL) sport fish contaminant residues were studied in human blood and urine specimens from 32 sport fish consumers from three Great Lakes: Lake Michigan (n = 10), Lake Huron (n = 11), and Lake Erie (n = 11). Serum was analyzed for 8 polychlorinated dioxin congeners, 10 polychlorinated furan congeners, 4 coplanar and 32 other polychlorinated biphenyl (PCB) congeners, and 11 persistent chlorinated pesticides. Whole blood was analyzed for mercury and lead. Urine samples were analyzed for 10 nonpersistent pesticides (or their metabolites) and 5 metals. One individual was excluded from statistical analysis because of an unusual exposure to selected analytes. Overall, the sample (n = 31) consumed, on average, 49 GL sport fish meals per year for a mean of 33 years. On average, the general population in the GL basin consume 6 meals of GL sport fish per year. The mean tissue levels of most persistent, bioaccumulative compounds also found in GL sport fish ranged from less than a twofold increase to that of PCB 126, which was eight times the selected background levels found in the general population. The overall mean total toxic equivalent for dioxins, furans, and coplanar PCBs were greater than selected background levels in the general population (dioxins, 1.8 times; furans, 2.4 times; and coplanar PCBs, 9.6 times). The nonpersistent pesticides and most metals were not identified in unusual concentrations. A contaminant pattern among lake subgroups was evident. Lake Erie sport fish consumers had consistently lower contaminant concentrations than consumers of sport fish from Lake Michigan and Huron. These interlake differences are consistent with contaminant patterns seen in sport fish tissue from the respective lakes; GL sport fish consumption was the most likely explanation for observed contaminant levels among this sample. Frequent consumers of sport fish proved to be effective sentinels for identifying sport fish contaminants of concern. In the larger study to follow, serum samples will be tested for PCBs (congener specific and coplanar), DDE, dioxin, and furans.     

Dioxin-like and non-dioxin-like toxic effects of polychlorinated biphenyls (PCBs): implications for risk assessment

Polychlorinated biphenyls (PCBs) are persistent, bioaccumulative, and toxic contaminants in the environment. Individual PCB congeners exhibit different physicochemical properties and biological activities that result in different environmental distributions and toxicity profiles. The variable composition of PCB residues in environmental matrices and their different mechanisms of toxicity complicate the development of scientifically based regulations for the risk assessment. In this article various approaches for the assessment of risks of PCBs have been critically examined. Recent developments in the toxic equivalency factor (TEF) approach for the assessment of toxic effects due to dioxin-like PCBs have been examined. PCB exposure studies that describe non-dioxin-like toxic effects, particularly neurobehavioral effects and their effective doses in animals were compiled. A comparative assessment of effective doses for dioxin-like and non-dioxin-like effects by PCBs has been made to evaluate the relative significance of non-ortho-and ortho-substituted PCBs in risk assessment. Using mink as an example, relative merits and implications of using TEF and total PCB approaches for assessing the potential for toxic effects in wildlife was examined. There are several advantages and limitations associated with each method used for PCB risk assessment. Toxic effects due to coplanar PCBs occur at relatively smaller concentrations than those due to non-dioxin-like PCBs and therefore the TEF approach derives the risk assessment of PCBs, in the environment. The need for the refinement of TEF approach for more accurate assessment of risks is discussed.     

Species and organ dependence of PCB contamination in fish, foxes, roe deer, and humans

According to previous experimental results, PCBs are deposited in muscle fat in animals and in humans, although they also reach the brain, the liver, and the lungs. The aim of the present study was to determine the concentrations of the so-called "indicator PCBs" (PCB nos. 28, 52, 101, 138, 153, 180), as described by the German ordinance for maximum concentrations of contaminants in foodstuffs, in muscle tissue, liver, and brain of four different species: fish, fox, roe deer, and humans, all exposed to PCBs directly in their environment. Potential target organs for the accumulation of these congeners were also to be identified. Furthermore, the organs or tissues were to be identified in which PCBs are accumulated, and unusual patterns of accumulation or breakdown of particular PCBs, for example the "dioxin-like PCBs" (coplanar PCBs) determined. For humans, the lungs were also included in these studies. Statistical comparison of PCB concentrations in samples from wild animals and humans showed that in spite of its relatively high fat concentration, brain tissue in all of the species examined (with the exception of fish) appeared to be better protected against accumulation of PCB than liver or muscle tissue. This protection may be the result of the blood-brain barrier, as witnessed by the relatively uniform concentration of PCBs throughout the various organs of fish, since the blood-brain barrier of fish is considerably less efficient than that of mammals. No peculiarities were found in regard to distribution of the coplanar PCBs over the other congeners in this study. This applies to the brain and other organs or tissues of the four species that were examined. Accumulations of PCBs and coplanar PCBs in the liver were only found in fox and roe deer. In contrast, humans were found to have accumulations of the high-chlorinated biphenyls studied here as well as PCB no. 118 in muscle tissue fat and not in the liver. Unexpectedly, low-chlorinated biphenyls were found to accumulate in the human lung. The results of this study show that the lung represents a target organ for the accumulation of potentially metabolically activated low-chlorinated biphenyls, indicating that the possible biological effects of PCBs on the lungs will need to receive more attention in the future.     

Modification by polychlorinated biphenyls (PCBs) of cadmium induced lesions in the planarian model, Dugesia dorotocephala

The appearance of abnormal growths on the planarian, Dugesia dorotocephala, in response to cadmium with and without pre-exposure to L-buthionine-R, S-sulfoximine (BSO) and concurrent exposure to the polychlorinated biphenyls (PCBs) Aroclor 1254, PCB 28, PCB 110 or PCB 126 is described. Pigmented rose thorn (PRT) lesions were non-invasive and appeared in response to PCBs. Post-head (PH) lesions developed in up to 100% of the animals within 6-20 days post-dosing, progressed rather rapidly and were highly invasive. Round tail tip (RTT) lesions appeared in lower frequencies within 10-30 days, but progressed extremely rapidly resulting in tail loss within 48 h. We have referred to these types of lesions as "tumors", but they are not necessarily characteristic of vertebrate neoplasms. PCBs interacted with cadmium in a complex way, in some cases increasing total lesions and decreasing time-to-lesion and in other cases having the opposite effects. A three-factor (PCB, PCB dose, Cd dose) nested analysis of variance model was used to determine lesion rates in order to compare PCB potencies as potentiators or antagonists. The Aroclor mixture was always the least potent co-toxicant but appeared to be the most potent antagonist; the coplanar PCB 126 was the most potent co-toxicant. The complex response surfaces and the lack of stoichiometry in dose-response relationships indicate that multiple mechanisms are responsible for PH and RTT lesions in planarians. These results emphasize the complexity of PCB toxicities and suggest further studies to validate the planarian model as a screen for combinations or environmental mixtures which may have altered biological potency in other species.     

Altered Gq/G11 guanine nucleotide regulatory protein expression in a rat model of hepatocellular carcinoma: role in mitogenesis

Guanine nucleotide regulatory proteins (G-proteins) represent an important transmembrane pathway whereby extra-cellular signals are transduced to intracellular signaling pathways. The mitogen-activated protein kinase (MAPK) cascade has been identified as a key factor in transducing numerous mitogenic stimuli. MAPK activity is regulated via numerous receptor types, including those linked to Gq/G11-proteins, which regulate phospholipase-C activity. We hypothesized that alterations in a Gq/G11-PLC pathway may contribute to the enhanced cellular mitogenesis characteristic of hepatocellular carcinoma (HCC), possibly via a MAPK-dependent pathway. By using an in vivo model of HCC we investigated changes in Gq/G11-protein expression in tumorigenic tissue versus adjacent, non-neoplastic liver. In addition we addressed the role of Gq/G11-proteins in the regulation of MAPK-linked mitogenesis by using rat hepatic tumorigenic cells (H4IIE) and isolated hepatocytes in culture. Western blot analysis showed significant increases in Gqalpha and G11alpha expression in tumorigenic liver versus normal liver specimens, an effect that was augmented in cultured H4IIE cells versus isolated cultured hepatocytes. Furthermore, phosphoinositol specific phospholipase-C (PLC) activity was significantly increased in HCC versus normal liver. A specific PLC inhibitor (Et-18-OCH3) caused a dose-dependent decrease in serum stimulated DNA synthesis in both cultured H4IIE cells and isolated rat hepatocytes, the H4IIE cell line showing greater sensitivity to Et-18-OCH3. In addition, serum-stimulated MAPK activity was significantly enhanced in H4IIE versus cultured hepatocytes. Moreover, treatment with Et-18-OCH3 significantly attenuated serum stimulated MAPK activity in both cultured hepatocytes and H4IIE cells. Furthermore, U73122 (Gqalpha-PLC specific uncoupler) and GP2A (Gqalpha specific inhibitor) mirrored the effects of those observed for Et-18-OCH3 whereas PD98059 (specific MEK inhibitor) completely abolished serum-stimulated DNA synthesis in tumorigenic H4IIE cells. We conclude that HCC is associated with enhanced Gq/G11-PLC expression/activity as compared with normal liver. Furthermore, a PLC-linked MAPK cascade plays a significant role in the progression of the enhanced mitogenesis characteristic of HCC.     

A comparison of dioxins, dibenzofurans and coplanar PCBs in uncooked and broiled ground beef, catfish and bacon

The primary source of dioxins (PCDDs), dibenzofurans (PCDFs) and coplanar PCBs for the general population is food, especially meat, fish, and dairy products. However, most data on the levels of these chemicals is from food in the raw or uncooked state. We report here the effect of one type of cooking (broiling) on the levels of PCDDs, PCDFs, and coplanar PCBs in ground beef (hamburger), bacon and catfish. Samples of hamburger, bacon, and catfish were broiled and compared to uncooked samples in order to measure changes in the amounts of dioxins in cooked food. The total amount of PCDD, PCDF, and coplanar PCB TEQ decreased by approximately 50% on average for each portion as a result of broiling the hamburger, bacon and catfish specimens. The mean concentration (pg TEQ/kg, wet weight) of PCDDs, PCDFs, and coplanar PCBs, however, remained the same in the hamburger, increased by 83% in the bacon, and decreased by 34% in the catfish. On average, the total measured concentration (pg/kg) of the congeners of PCDDs, PCDFs, and coplanar PCBs increased 14% in the hamburger, increased 29% in the bacon, and decreased 33% in the catfish.     

Polychlorinated diphenyl ethers, dibenzo-p-dioxins, dibenzofurans and biphenyls in seals and sediment from the Gulf of Finland

Polychlorinated diphenyl ethers (PCDEs), 2,3,7,8-substituted polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) were analyzed in seals from the Gulf of Finland and in sediments from the Gulf of Finland and near Gotland. The sediments included one surface core from both sampling sites. The seal material consisted of 14 ringed seals and 6 grey seals that all were found dead and examined for pathology. The main aims were to scrutinize levels and patterns of PCDEs for the first time in seals from the Baltic Sea and to estimate whether chlorinated compounds mentioned have an influence on an exceptional high mortality that occurred among ringed seals in the Gulf of Finland in late 1991. The concentrations of 50 congeners of tetra- through deca-CDEs analyzed ranged from < 0.3 to 62 ng/g lipid weight (lw) in seal blubber, but in the sediments PCDEs were non-detectable (tetra- through hepta-CDEs < 0.1 ng/g dry weight (dw)). In ringed seals with good nutritional status, the concentrations of almost all PCDE congeners were greater in two adult females than in specimens of younger age groups. The concentrations of PCDDs and PCDFs as TCDD-equivalents exceeded those of the coplanar (non-ortho) PCBs in sediments, whereas non- and monoortho PCBs constituted greater toxic loads as those calculated for PCDDs and PCDFs in seals. However, the levels revealed do not explain the high mortality of ringed seals.     

Decrease in levels and body burden of dioxins, dibenzofurans, PCBS, DDE, and HCB in blood and milk in a mother nursing twins over a thirty-eight month period

This paper presents measured dioxin, dibenzofuran, PCB, DDE and HCB blood and milk levels and estimated body burdens in a mother who nursed twins for thirty-eight months. A total of thirteen milk samples and three blood samples were collected and analyzed. Measured PCDD and PCDF levels in milk decreased from 309 and 21 ng/kg (ppt) to 173 and 9 ng/kg, respectively, between March 1993 and September 1995. Based on the decrease in breast milk dioxin levels, we estimate that the nursing mother reduced her dioxin body burden from 310 to 96 ng dioxin toxic equivalents (TEQs), or approximately 69%. In two and one half years the level of HCB in the mother's milk decreased from 10.7 to less than 1.8 ng/g (ppb), the level of DDE decreased from 246 to 46 ng/g and the total level of non-coplanar PCBs decreased from 285 to 63 ng/g, on a lipid basis. We estimate that the twin's consumption of dioxins, dibenzofurans, and coplanar PCBs from breast feeding was approximately 115 ng TEQ per twin.     

Toxic equivalency factors (TEFs) for PCBs, PCDDs, PCDFs for humans and wildlife

An expert meeting was organized by the World Health Organization (WHO) and held in Stockholm on 15-18 June 1997. The objective of this meeting was to derive consensus toxic equivalency factors (TEFs) for polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) and dioxinlike polychlorinated biphenyls (PCBs) for both human, fish, and wildlife risk assessment. Based on existing literature data, TEFs were (re)evaluated and either revised (mammals) or established (fish and birds). A few mammalian WHO-TEFs were revised, including 1,2,3,7,8-pentachlorinated DD, octachlorinated DD, octachlorinated DF, and PCB 77. These mammalian TEFs are also considered applicable for humans and wild mammalian species. Furthermore, it was concluded that there was insufficient in vivo evidence to continue the use of TEFs for some di-ortho PCBs, as suggested earlier by Ahlborg et al. [Chemosphere 28:1049-1067 (1994)]. In addition, TEFs for fish and birds were determined. The WHO working group attempted to harmonize TEFs across different taxa to the extent possible. However, total synchronization of TEFs was not feasible, as there were orders of a magnitude difference in TEFs between taxa for some compounds. In this respect, the absent or very low response of fish to mono-ortho PCBs is most noticeable compared to mammals and birds. Uncertainties that could compromise the TEF concept were also reviewed, including nonadditive interactions, differences in shape of the dose-response curve, and species responsiveness. In spite of these uncertainties, it was concluded that the TEF concept is still the most plausible and feasible approach for risk assessment of halogenated aromatic hydrocarbons with dioxinlike properties.     

Effects of amino acids on alcohol intake in stroke-prone spontaneously hypertensive rats

The concentration-dependent effects of several PCB, PCDD, and PCDF congeners and several commercial PCB preparations as antiestrogens were determined in the aryl hydrocarbon (Ah)-responsive MCF-7 human breast cancer cell lines. The inhibition of the 17 beta-estradiol-induced secretion of the 52-kDa protein (procathepsin D) was measured using a combination of polyacrylamide gel electrophoresis, double-staining of the protein bands with ISS ProBlue and silver stain, and quantitation by densitometric analysis. For the PCBs, the order of antiestrogenic potency was 3,3',4,4',5-pentachlorobiphenyl > 3,3',4,4',5,5'-hexachlorobiphenyl approximately 3,3',4,4'-tetrachlorobiphenyl > 2,3,3',4,4',5'-hexa, 2,3,3',4,4'- and 2,3,4,4',5-pentachlorobiphenyl > Aroclors 1221, 1232, 1248, 1254, and 1260 were inactive as antiestrogens at the highest concentrations used in this study (10(-6) M). For the PCDDs and PCDFs, the order of antiestrogenic potency was 2,3,7,8-tetrachlorodibenzo-p-dioxin > 2,3,7,8-tetrachlorodibenzofuran > 2,3,4,7,8-pentachlorodibenzofuran > 1,2,3,7,9-pentachlorodibenzofuran > 1,3,6,8-tetrachlorodibenzofuran. With few exceptions, the order of potency for all these congeners and mixtures paralleled their relative activities as agonists for other Ah receptor-mediated responses and their competitive binding affinities for the Ah receptor. The results of this study support the role for the Ah receptor in mediating the inhibition of the 17 beta-estradiol-induced secretion of the 52-kDa protein in MCF-7 cells and also points out the utility of this technique as a bioassay for this class of compounds.     

Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene

Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, and recently bioassay-based induction studies have been used to determine exposures to complex mixtures of PAHs. Induction of CYP1A1-dependent activity in H4IIE rat hepatoma cells has been used extensively as a bioassay for halogenated aromatic hydrocarbons and more recently for PAHs. Fluoranthene (FL) is a prevalent PAH contaminant in diverse environmental samples, and FL did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity significantly in H4IIE cells. However, in cells cotreated with 2 x 10(-5) M FL plus the potent inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[k]fluoranthene (BkF) (2 x 10(-8) M), there was a significant decrease in EROD activities. Furthermore, treatment of TCDD-induced rat microsomes with FL caused an 80% decrease in EROD activity. Studies showed that FL did not affect induction of CYP1A1 protein or mRNA levels in H4IIE cells, and analysis of enzyme inhibition data using microsomal CYP1A1 indicated that FL noncompetitively inhibited CYP1A1-dependent activity. 32P-Postlabeling revealed no significant FL-DNA adduct formation in H4IIE cells treated with FL. However, in cells cotreated with FL plus BkF or benzo[a]pyrene (BaP), certain PAH-DNA adducts were induced 2-fold. This study demonstrated that FL is an inhibitor of CYP1A1-dependent enzyme activity in rat hepatoma H4IIE cells and that the genotoxic potency of some carcinogenic PAHs may be modulated by FL in mixtures containing relatively high levels of this compound.     

3,4,3',4'-Tetrachlorobiphenyl acts as an estrogen in vitro and in vivo

Polychlorinated biphenyls (PCBs) are one of the most widespread, persistent man-made products in the ecosystem giving rise to serious environmental contamination and potential hazard to health. The PCBs, in common with other compounds such as the dioxins, have been shown to exert some biological actions mediated through the aryl hydrocarbon receptor. Evidence for interaction of PCBs with other nuclear receptors has been sparse. Here we present evidence that 3,4,3',4'-tetrachlorobiphenyl (TCB) (PCB77), a PCB with high toxicity and significant bioaccumulation, can act as an estrogen with actions mediated through the estrogen receptor. Evidence is presented from multiple assay systems including 1) ligand binding to estrogen receptor in a competitive binding assay, 2) ligand ability to induce estrogen receptor binding to DNA, 3) ligand regulation of gene expression from a transfected exogenous (ERE-tk-CAT) or an endogenous (pS2) estrogen-regulated gene, 4) ligand regulation of cell growth in estrogen-dependent human breast cancer cell lines MCF7 and ZR-75-1, and 5) ligand activity in the immature mouse uterine weight bioassay in vivo. These results demonstrate that TCB (PCB77) can be included in the increasing list of environmental pollutants that possess the ability to mimic estrogen action and be termed an environmental estrogen. Since the concentrations of TCB used here (10(-9) M; 292 ng/liter) are not incompatible with levels of PCB/TCB found in human tissues, these results may have physiological relevance. Use of multiple approaches to study estrogenic action demonstrates that one congener can act as both an agonist and antagonist of estrogen action and that the magnitude of these effects can alter according to the molecular environment.     

Effects of in vitro exposure of beluga whale leukocytes to selected organochlorines

The effects of in vitro exposure to different organochlorines were evaluated on immune functions of beluga whale peripheral blood leukocytes and splenocytes. The effects of different concentrations of four different congeners of PCBs (138, 153, 180, and 169) as well as two DDT metabolites (p,p'-DDT and p,p'-DDE) were evaluated on phagocytosis and cell proliferation. The effects of dioxins and mixtures of organochlorines were also evaluated on cell proliferation. The different compounds tested had no marked effect on phagocytosis. PCB 138 and p,p'-DDT, but not PCB 153, PCB 180, PCB 169, and p,p'-DDE, reduced significantly the proliferative response of beluga splenocytes cultured either with or without phytohemagglutinin A (PHA). Proliferation of beluga splenocytes was not markedly affected by exposure to 5 ppm of PCB 138, 153, 180, and 169 separately. Exposure to a mixture of congeners 138, 153, and 180 (5 ppm each) significantly reduced splenocytes proliferation, but not the mixture of congeners 138, 153, 180, and 169 (5 ppm each). TCDD did not affect cell proliferation in our study. The reduced proliferation of beluga cells exposed in vitro to mixtures of organochlorines at concentrations in the range of those observed in tissues of St. Lawrence belugas might provide a basis to support the hypothesis that contaminants induce immunosuppression in these animals.     

The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [3H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [3H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a Kd of 113 nM and Bmax of 0.65 pmol/10(6) cells in freshly isolated cell suspension and Kd of 1.65 muM and Bmax of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [3H]PAF itself was found in the same cell preparations. The binding of [3H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K(i) of 3.8 nM and Bmax of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam was competed for the [3H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [3H]WEB 2086 with a rank order BN 50739>Ro 5-4864 > or = clonazepam. Interestingly, bicuculline, specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [3H]WEB 2086. The binding of [3H]flunitrazepam was inhibited with a rank potency BN 50739>WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

Novel formula for cell kinetics in xenograft model of hepatocellular carcinoma using histologically calculable parameters

Hepatocellular carcinoma (HCC) is associated with increased expression and function of inhibitory guanine nucleotide regulatory proteins (Gi-proteins). This study addresses the effects of chronic ethanol exposure on the expression and function of adenylyl cyclase (AC)-linked G-proteins (Gs and Gi) and growth in experimental HCC. G-protein expression and function was determined by immunoblot in the hepatic tumorigenic H4IIE cell line and isolated cultured hepatocytes in the absence or presence of ethanol (5-100 mmol/L). Chronic exposure (24 hours) to ethanol dose-dependently increased Gialpha1/2 expression in the H4IIE cell line, but not in cultured hepatocytes. Gsalpha-protein expression remained unchanged in both H4IIE cells and cultured hepatocytes following ethanol treatment. In addition, ethanol directly activated a Gi-protein, because pertussis toxin (PTx)-catalyzed, adenosine diphosphate (ADP)-dependent ribosylation of Gialpha substrates decreased following ethanol treatment. The increased functional activity of Gialpha1/2-protein expression was confirmed by demonstrating that ethanol dose-dependently inhibited basal and stimulated AC activity in H4IIE cells, while not significantly altering basal AC activity in isolated cultured hepatocytes. Furthermore, while ethanol had no significant effect on basal mitogenesis in H4IIE cells or hepatocytes, increased mitogenesis caused by direct Gialpha-protein stimulation (mastoparan M7; 10-5,000 nmol/L) was further enhanced in the presence of ethanol, an effect that was completely blocked following Gi-protein inhibition (PTx; 100 ng/mL). In contrast, activation of Gi-proteins using M7 failed to alter cellular mitogenesis in isolated cultured hepatocytes, whether in the absence or presence of ethanol. Finally, analysis of mitogen-activated protein kinase (MAPK) activity demonstrated that chronic ethanol treatment further enhanced Gi-protein-stimulated MAPK activity in hepatic tumorigenic cells. In conclusion, these data demonstrate that ethanol enhances cellular mitogenesis in experimental HCC as a result of, at least in part, a Gi-MAPK-dependent pathway. Furthermore, this effect may be caused by ethanol's direct up-regulation of the expression and activity of Gi-proteins in HCC.     

A survey of PCB congeners in U.K. cows' milk

Samples of unpasteurised bulked milk, taken directly from ten herds of dairy cattle on rural and urban farms in the north west of England on five separate sampling occasions, were analysed for a range of PCB congeners. Sigma PCB concentrations (sum of 37 congeners) ranged from 3.4-16.4 ng/g milk fat with a mean sigma PCB concentration of 8.4 ng/g milk fat. The dominating congeners were 118, 153, 138 and 180, which contributed 15%, 20%, 17% and 9% of the sigma PCB load respectively. The chlorine pattern of the congeners which made moderate or major contributions to the sigma PCB concentration were typically substituted at both para positions (4,4'), while the PCB congeners not detected in the milk had at least one ring that was not 4-substituted. These results indicate the 4,4' substitution pattern as being the key to PCB persistence in cows. It is estimated that consumption of typical daily intakes of milk with the PCB concentrations measured in this study would contribute 11% of the average daily sigma PCB intake for individuals in the UK. This contribution would increase to 30% when exposure through the consumption of dairy products prepared from such milk (e.g. cheese, butter) is taken into account. It is estimated that the inclusion of the TEF assigned PCBs would typically increase the TEQ rating of cows' milk by approximately 40% over that attributed to PCDD/Fs alone.