Predictors of serum polychlorinated biphenyl concentrations in Anniston residents.

The Anniston Community Health Survey was a community-based cross-sectional study of Anniston, Alabama, residents who live in close proximity to a former PCB production facility to identify factors associated with serum PCB levels. The survey comprises 765 Anniston residents who completed a questionnaire interview and provided a blood sample for analysis in 2005-2007. Several reports based on data from the Anniston survey have been previously published, including associations between PCB exposure and diabetes and blood pressure. In this study we examine demographic, behavioral, dietary, and occupational characteristics of Anniston survey participants as predictors of serum PCB concentrations. Of the 765 participants, 54% were White and 45% were African-American; the sample was predominantly female (70%), with a mean age of 55 years. Serum PCB concentrations varied widely between participants (range for sum of 35 PCBs: 0.11-170.4 ng/g wet weight). Linear regression models with stepwise selection were employed to examine factors associated with serum PCBs. Statistically significant positive associations were observed between serum PCB concentrations and age, race, residential variables, current smoking, and local fish consumption, as was a negative association with education level. Age and race were the most influential predictors of serum PCB levels. A small age by sex interaction was noted, indicating that the increase in PCB levels with age was steeper for women than for men. Significant interaction terms indicated that the associations between PCB levels and having ever eaten locally raised livestock and local clay were much stronger among African-Americans than among White participants. In summary, demographic variables and past consumption of locally produced foods were found to be the most important predictors of PCB concentrations in residents living in the vicinity of a former PCB manufacturing facility.

Serum concentrations of polychlorinated biphenyls (PCBs) in participants of the Anniston Community Health Survey.

Serum concentrations of 35 ortho-substituted polychlorinated biphenyl congeners (PCBs) were measured in 765 adults from Anniston, Alabama, where PCBs were manufactured between 1929 and 1971. As part of the Anniston Community Health Survey (ACHS), demographic data, questionnaire information, and blood samples were collected from participants in 2005-2007. Forty-six percent of study participants were African-American, 70% were female, and the median age was 56 years. The median concentration of the sum of 35 PCB congeners (ΣPCBs) was 528 ng/g lipid, with a 90th percentile of 2,600 ng/g lipid, minimum of 17.0 ng/g lipid, and maximum of 27,337 ng/g lipid. The least square geometric mean ΣPCBs was more than 2.5 times higher for African-American participants than for White participants (866 ng/g lipid vs. 331 ng/g lipid); this difference did not change materially after adjustment for age, sex, body mass index (BMI) and current smoking. In spite of large differences in absolute PCB levels, relative contributions of individual congeners to ΣPCBs were quite similar between race groups. Nevertheless, while percent contributions to ΣPCBs for most of the most abundant penta- to heptachlorobiphenyls were higher among African-Americans, the percentages were higher in Whites for the lower-chlorinated PCBs 28 and 74 and for octa- to decachlorinated PCBs. No major differences were observed in geometric mean ΣPCBs between women and men when adjusted for age, race, BMI and current smoking (516 ng/g lipid vs. 526 ng/g lipid). Principal component analysis revealed groups of co-varying congeners that appear to be determined by chlorine substitution patterns. These congener groupings were similar between ACHS participants and the National Health and Nutrition Examination Survey (NHANES) 2003-04 sample of the general United States population, despite ACHS participants having serum concentrations of ΣPCBs two to three times higher than those in comparable age and race groups from NHANES.

Fabrication and mechanical properties of PLLA/PCL/HA composites via a biomimetic, dip coating, and hot compression procedure.

Currently, the bone-repair biomaterials market is dominated by high modulus metals and their alloys. The problem of stress-shielding, which results from elastic modulus mismatch between these metallic materials and natural bone, has stimulated increasing research into the development of polymer-ceramic composite materials that can more closely match the modulus of bone. In this study, we prepared poly(L: -lactic acid)/hydroxyapatite/poly(epsilon-caprolactone) (PLLA/HA/PCL) composites via a four-step process, which includes surface etching of the fiber, the deposition of the HA coating onto the PLLA fibers through immersion in simulated body fluid (SBF), PCL coating through a dip-coating process, and hot compression molding. The initial HA-coated PLLA fiber had a homogeneous and continuous coating with a gradient structure. The effects of HA: PCL ratio and molding temperature on flexural mechanical properties were studied and both were shown to be important to mechanical properties. Mechanical results showed that at low molding temperatures and up to an HA: PCL volume ratio of 1, the flexural strain decreased while the flexural modulus and strength increased. At higher mold temperatures with a lower viscosity of the PCL a HA: PCL ratio of 1.6 gave similar properties. The process successfully produced composites with flexural moduli near the lower range of bone. Such composites may have clinical use for load bearing bone fixation.

Influence of pretreatment on the surface characteristics of PLLA fibers and subsequent hydroxyapatite coating.

A fibrous precursor for bone repair composites was made by coating poly(L-lactide) (PLLA) fibers with hydroxyapatite (HA) using a biomimetic method. To enhance the bonding between the HA coating and the PLLA fiber, PLLA fibers were etched with either sodium hydroxide or sodium hypochlorite to generate carboxyl groups on fiber surfaces. The experiments were designed to determine the influence of etching on the fiber surface morphology and chemistry as well as the subsequent HA coating on the etched fiber surfaces. It was found that the etching pretreatment increased the roughness as well as the hydrophilicity of fibers, indicating that hydrolysis of PLLA chains had taken place on fiber surfaces. The etching pretreatment also promoted HA coating formation by introducing thicker coating on the surface of fibers with a longer etching time, a higher etching concentration, or with NaOCl as the etching agent. A mechanism of surface hydrolysis and oxidation of PLLA was proposed.

Effect of TCDD exposure on CYP1A1 and CYP1B1 expression in explant cultures of human endometrium.

Endometriosis is a debilitating disease estimated to affect 10% of reproductive-age women and characterized by the growth of endometrial tissue outside of the uterus. The present study characterizes a human endometrial explant culture model for studying the direct effects of TCDD exposure by assessing the expression of CYP1A1 and CYP1B1 mRNA (Northern blotting), protein (Western blotting), and activity (7-ethoxyresorufin-O-deethylase; EROD) in explants cultured with and without TCDD. Explants were obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. The explants were cultured with 10 nM estradiol (E(2)) or 1 nM E(2) plus 500 nM progesterone (P(4)) with or without TCDD (first 24 h). The expression of CYP1A1 and CYP1B1 mRNA was greatest with 10 nM TCDD and increased up to 72 h after initial exposure. EROD activity increased up to 120 h. Explants from a secretory phase biopsy became reorganized in culture and formed a new epithelial membrane, while maintaining basic endometrial morphology and viability for up to 120 h. At 24 h, TCDD significantly increased CYP1A1 and CYP1B1 mRNA, and at 72 h, TCDD significantly increased EROD activity and CYP1B1 protein compared to explants cultured without TCDD for similar times. CYP1B1 protein also exhibited substantial constitutive expression that was similar in uncultured biopsies, where CYP1B1 protein was immunolocalized in the cytoplasm of epithelial glands, with only occasional patches of protein in the surface epithelial membrane. In explants cultured with and without TCDD exposure, CYP1B1 protein was localized in the cytoplasm of the new surface epithelial membrane and glands closest to the surface. CYP1A1 protein was not detected in uncultured biopsies or explants. Both younger age (age 30 and under) and proliferative phase were associated with higher TCDD-induced EROD activity in specimens treated with E(2):P(4). No significant endometriosis-related differences were observed for any of the biomarkers, but the detection of disease-specific change was limited by small sample size and variability in tissue-cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase and hormonal exposure.

Metabolism of benzo(a)pyrene by duck liver microsomes.

The metabolism of benzo(a)pyrene [BP], a model carcinogenic PAH, by hepatic microsomes of two duck species, mallard (Anas platyrhynchos) and common merganser (Mergus merganser americanus) collected from chemically-contaminated and relatively non-contaminated areas was investigated. The rate of metabolism of BP by liver microsomes of common merganser and mallard collected from polluted areas (2,650 +/- 310 and 2,200 +/- 310 pmol/min per mg microsomal protein, respectively) was significantly higher than that obtained with liver microsomes of the two species collected from non-polluted areas (334 +/- 33 and 231 +/- 30 pmol/min per mg microsomal protein, respectively). The level of cytochrome P-450 1A1 was significantly higher in the liver microsomes of both duck species from the polluted areas as compared to the ducks from the non-polluted areas. The major BP metabolites, including BP-9, 10-diol, BP-4, 5-diol, BP-7, 8-diol, BP-1, 6-dione, BP-3, 6-dione, BP-6, 12-dione, 9-hydroxy-BP and 3-hydroxy-BP, formed by liver microsomes of both duck species from polluted and non-polluted areas, were qualitatively similar. However, the patterns of these metabolites were considerably different from each other. Liver microsomes of ducks from the polluted areas produced a higher proportion of benzo-ring dihydrodiols than the liver microsomes of ducks from the non-polluted areas, which converted a greater proportion of BP to BP-phenols. The predominant enantiomer of BP-7,8-diol formed by hepatic microsomes of the two duck species had an (-)R,R absolute stereochemistry. The data suggest that duck and rat liver microsomal enzymes have different regioselectivity but similar stereoselectivity in the metabolism of BP.

Time- and concentration-dependent induction of CYP1A1 and CYP1A2 in precision-cut rat liver slices incubated in dynamic organ culture in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

In a previous 24-h study, precision-cut rat liver slices were validated as a useful in vitro model for assessing the dose-related induction of CYP1A1 and CYP1A2 in rat liver following exposure to 2, 3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Further assessment of the utility of this model was accomplished by initially exposing rat liver slices to medium containing TCDD (0.01 nM) for 24 h and incubating the slices up to an additional 72 h in TCDD-free medium. The slices remained viable throughout the incubation period with an intracellular potassium content varying from 45.2 +/- 2.3 micromol/g at 48 h to 50.0 +/- 1.6 micromol/g at 72 h. In TCDD-exposed slices, CYP1A1 protein and its respective enzymatic activity, the O-deethylation of ethoxyresorufin (EROD), significantly increased with time over the 96-h incubation period, with EROD activity increasing from 63.6 +/- 14.2 at 24 h to 905 +/- 291 pmol/mg/min at 96 h. Under identical incubation conditions, but in the absence of TCDD, the EROD activity for the control liver slices ranged from 14. 3 +/- 4.3 to 44.9 +/- 11.9 pmol/min/mg. Conversely, the level of CYP1A2 protein and its respective activity (acetanilide hydroxylation) transiently decreased from 24 to 96 h with no significant differences observed between the control (0 nM TCDD) and treatment group (0.01 nM TCDD). The concentration-effect relationship at 96 h was characterized by incubating rat liver slices for the initial 24 h in medium containing TCDD at concentrations ranging from 0.1 pM to 10 nM. Induction of CYP1A1 protein and EROD activity was observed for all treatment groups with the 10 nM TCDD treatment group displaying greater than 100-fold induction compared to control (0 nM TCDD). Immunohistochemical localization of CYP1A1 protein within liver slices supported the time- and concentration-dependent induction of EROD activity by TCDD. The induction of CYP1A1 was initially observed to be centrilobular, with increased expression due to both elevated CYP1A1 within cells and the recruitment of additional cells expressing CYP1A1 throughout the entire liver slice. Additionally, the immunohistochemical analysis of the liver slices demonstrated the conservation of tissue architecture following up to 96 h of incubation in dynamic organ culture and provided further evidence for maintenance of tissue viability. In comparison to CYP1A1, the induction of CYP1A2 at 96 h was a less sensitive response, with significant induction of CYP1A2 protein and its respective activity occurring at a medium concentration of 0.1 nM TCDD (686 pg/g liver). In general, increasing the incubation period from 24 to 96 h markedly increased TCDD-induced expression of CYP1A1 and minimally enhanced CYP1A2 expression. Moreover, extending the incubation period to 96 h resulted in in vitro induction profiles for CYP1A1 and CYP1A2 that were qualitatively and quantitatively similar to that previously observed following in vivo exposure to TCDD (Drahushuk et al., Toxicol. Appl. Pharmacol. 140, 393-403, 1996).

Detection of CYP1A1 protein in human liver and induction by TCDD in precision-cut liver slices incubated in dynamic organ culture.

Cytochrome P4501A1 (CYP1A1) has been implicated in the conversion of numerous polycyclic aromatic hydrocarbons into electrophilic species capable of binding covalently to DNA and has therefore been postulated to be involved in the initiation of carcinogenesis. The expression of CYP1A1 protein appears not to be constitutive, but is readily inducible by aryl hydrocarbon (Ah) receptor ligands in a majority of tissues of experimental animals, especially the liver. To date, there is conflicting evidence for the expression or inducibility of CYP1A1 protein in human liver. In this present study, we report the detection of CYP1A1 in all 20 human liver microsomal samples tested by standard western immunoblotting with chemiluminescent detection using a specific monoclonal antibody (mAb 1-12-3) directed against a marine fish (scup) cytochrome P450E. mAb 1-12-3 has been shown previously to specifically recognize CYP1A1 in mammals. This system consistently demonstrated a detection sensitivity as low as 0.01-0.025 pmol CYP1A1 per lane. In the samples where CYP1A1 protein levels were quantitated, CYP1A1 ranged from approximately 0.4 to 5 pmol CYP1A1/mg microsomal protein. Additionally, the inducibility of CYP1A1 protein was demonstrated by incubating precision-cut human liver slices in dynamic organ culture for up to 96 h in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The specificity of mAb 1-12-3 was tested using several purified human and rat cytochrome P450s to ensure that the protein being detected was CYP1A1. mAb 1-12-3 did not cross-react with human CYP1A2 or CYP3A4 or rat CYP1B1, but did strongly recognize CYP1A1. However, there was a very weak cross-reactivity of mAb 1-12-3 with human CYP2E1, approximately 75-fold less compared with CYP1A1. In order to confirm CYP1A1 as the immunoreactive protein detected in human liver, microsomal samples were subjected to two-dimensional electrophoresis involving isoelectric focusing followed by SDS-PAGE and immunoblotting. Utilizing mAb 1-12-3, the human liver microsomal samples displayed an immunoblotting profile matching that obtained from a microsomal preparation from a AHH-1 TK+/- cell line expressing solely human CYP1A1 and differing from the profile obtained using a polyclonal antibody directed against CYP2E1 and cells expressing CYP2E1. Furthermore, mAb 1-12-3 recognized only one protein of identical mobility on the two-dimensional blots from human liver microsomes and AHH-1 TK+/- cells expressing CYP1A1, while displaying no reaction to cells expressing only CYP2E1. In conclusion, CYP1A1 appears to be expressed in human liver at low levels and is inducible upon exposure to TCDD.

Cytochrome P450 1A1 induction in rat lymphoid tissues following in vivo and in vitro exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin requires protein kinase C.

The induction of cytochrome P450 1A1 (CYP1A1) is one of the most sensitive responses associated with exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. The mechanisms that underlie this response are not completely understood, particularly in lymphoid tissues that may be used in biomarker studies in humans. CYP1A1 mRNA expression and enzyme activity (ethoxyresorufin-o-deethylase, EROD) were investigated in rat thymus and spleen and isolated thymocytes and splenocytes in culture. Thymus- or spleen-derived microsomes from rats treated in vivo with TCDD showed induced EROD activity after as little as 12 h following a single exposure to TCDD (5 microg/kg body weight). Resting rat thymocytes in culture had detectable levels of EROD activity and CYP1A1 mRNA expression which increased following in vitro exposure to > or = 0.1 nM TCDD for 24 or 48 h. Interestingly, concomitant in vitro exposure of rat thymocytes to TCDD and the mitogen concanavalin A (Con A) inhibited the induction of EROD activity, which is in contrast to the response of cultured human peripheral blood lymphocytes (Landi et al., 1994; Pharmacogenetics 4, 242 246). Resting rat splenocytes in culture had no detectable EROD activity and CYP1A1 activity could not be induced by in vitro TCDD exposure, in the presence or absence of Con A. These results suggest that the relative maturation state of the cells is important in regulating the expression of CYP1A1, since splenocytes represent a more mature population of B and T lymphocytes. TCDD-induced CYP1A1 expression in cultured rat thymocytes was inhibited by the addition of calphostin C, a specific protein kinase C (PKC) inhibitor, suggesting a role for PKC as a second messenger in the CYP1A1 induction pathway. In vivo co-exposure with phorbol-myristate-acetate (PMA) and TCDD also inhibited CYP1A1 induction. Again, suggesting a role for PKC in CYP1A1 induction. Together, these results indicate that relative lymphocyte maturation state and the PKC pathway are important factors in regulating the expression of CYP1A1.

Modulation of cytochrome P450 by 5,5'-bis-trifluoromethyl-2,2'-dichlorobiphenyl, a unique environmental contaminant.

5,5'-Bis-trifluoromethyl-2,2'-dichlorobiphenyl (5,5'CF3-2,2'PCB) is representative of a unique class of trifluoromethyl polychlorinated biphenyls (CF3-PCBs) found in sediments and fish of Lake Ontario, the Niagara river, and their tributaries. The potential hazard of 5,5'CF3-2,2'PCB was assessed by exposing male Wistar rats to this agent in corn oil at a dose of 1 mg/kg/day or 75 mg/kg/day or corn oil alone (control) by oral intubation for 7 consecutive days. No lethality occurred during the course of exposure. A significant increase in liver weight and liver/body weight ratio and significant decrease in body weight gain were observed following exposure to the high dose of CF3-PCB, relative to control. Exposure to the CF3-PCB also resulted in a dose-related increase in the total hepatic cytochrome P450 content. This was associated with a dose-related increase in the O-deethylation of ethoxycoumarin, an activity which is mediated by several cytochrome P450s and thus provides a general representation of cytochrome P450 status. Specifically, a dose-dependent induction of the cytochrome P450s 1A1 and 1A2 (CYP1A1 and CYP1A2) proteins and their respective activities was observed, with significant induction occurring in both the low and high dose CF3-PCB groups, compared to control. Additionally, CYP2B1 and CYP2B2 proteins and activities were induced following treatment with the high dose of 5,5'CF3-2,2'PCB. Since, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds selectively induce CYP1A1 and CYP1A2. the results suggest that 5,5'CF3-2,2'PCB has significant dioxin-like activity. Furthermore, 5,5'CF3-2,2'PCB appears to be acting as a mixed-type inducer since phenobarbital-like induction of CYP2B1 and 2B2 was also associated with exposure.

Considerations on genetic and environmental factors that contribute to resistance or sensitivity of mammals including humans to toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Part 1: Genetic factors affecting the toxicity of TCDD.

The marked species differences in short-term toxicity (30-day LD50) of ca. 10,000 (LD50: guinea pigs ca. 1 microgram/kg body wt and Han/Wistar Kuopio rats more than 9600 micrograms/kg body wt) of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the central issues of the controversies that have developed on the validity of risk assessment strategies for TCDD and related compounds. One of the most challenging issues that toxicologists face today is the identification of genes that contribute to or are responsible for increased resistance or sensitivity to TCDD and related compounds. It is assumed that most, if not all, toxic effects of TCDD are mediated more or less through the binding affinity to the Ah receptor. This hypothesis was extended and tries to explain the differences in sensitivity/resistance of animals including humans to TCDD by their total fat (lipid) content. In this respect the gene or genes which is or are responsible for obesity of mammals including humans are of great interest. An obvious linear positive logarithmic relationship between the oral 30-day LD50 (microgram/kg) of TCDD in different species and strains of mammals and their total body fat content (TBF%) was found: log LD50 = 5.30 x log (TBF)-3.22, or LD50 = 0.000603 x (TBF)5.30. By means of this regression the toxicity of TCDD in mammals including humans of different age and/or body weight can be predicted if their total body fat content is known. Examples of single-gene and polygenic disease models in different mammals, such as nonobese diabetic, diabetic, viable yellow, obese, and fat mice, as well as transgenic mice, and other suitable animal models, such as fatty Zucker rats, Han/Wistar (Kuopio) rats, and minipigs, are discussed, and predicted LD50 values of TCDD in these animals and humans are presented.

Levels of dioxins, dibenzofurans, PCB and DDE congeners in pooled food samples collected in 1995 at supermarkets across the United States.

Food, particularly dairy products, meat, and fish, has been identified as the primary immediate source of intake of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), and polychlorinated biphenyls (PCBs) for the general population. We previously reported PCDD/Fs in individual analyses of food samples from a number of countries, including the U.S., the former Soviet Union, and Vietnam. We also previously estimated daily intake of dioxins and related chemicals in Americans at various ages in these reports. In this paper, the levels of dioxins, dibenzofurans, dioxin toxic equivalents (TEQs), selected dioxin-like PCBs, and DDE (a persistent metabolite of DDT) were measured in 12 pooled food samples from over 90 individual specimens collected from supermarkets throughout the United States during 1995. Samples were pooled by food groups and then analyzed. Food samples were collected in Binghamton, New York; Atlanta, Georgia; Chicago, Illinois; San Diego, California; and Louisville, Kentucky. In addition to the meat, dairy, and fish samples, a vegan (all vegetable, fruit and grain, no animal product) diet, was simulated; this showed the lowest level of dioxins.

Cancer risk assessment using blood dioxin levels and daily dietary TEQ intake in general populations of industrial and non-industrial countries.

We previously measured polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) in U.S. foods and estimated the daily dioxin toxic equivalent (TEQ) intake to be from 0.3 to 3.0 pg 1-TEQ/kg body weight for adults. These values are similar to values reported in Canada, Germany, England, and the Netherlands. The U.S. Environmental Protection Agency (EPA) Dioxin Reassessment Draft Documents currently propose a cancer risk-specific dose estimate of 0.01 pg TEQ/kg body weight/day (U.S. EPA, 1994). This risk-specific dose estimate represents a lifetime (70 years) dose which results in a plausible upper bound cancer risk of 1 x 10(-6) (the probability of one additional cancer per one million exposed population over a lifetime). The proximate source of almost all dioxin intake in the general population is from food. Using our data for daily dietary dioxin exposure and the EPA's proposed risk specific dose, we estimate that over a lifetime a maximum of 30 to 300 excess cancers per million could result from the ingestion of dioxin containing food products. In the U.S. population of 260 million, a maximum range of 7,800 to 78,000 excess cancers over a lifetime (70 years) or 111 to 1,114 cases/year might be directly linked to dioxin exposure from food. Because these calculations are based on conservative approaches to setting an upper bound, "true" risk is not likely to exceed this value. At present, one-third of all Americans will develop cancer over a lifetime and one in four Americans are likely to die from cancer. For 1995, it was estimated that there would be 1,252,000 new cancer cases in the U.S. From this, we calculate that a maximum between 0.009% and 0.09% (111-1,114 cases/year) of all cancer cases in the U.S. might be directly linked to dioxin intake in food, assuming intakes have remained constant over a lifetime. Previous studies have suggested that as much as 70% of the total cancer risk in humans may be attributable to diet, primarily as a function of caloric and synthetic chemicals in the diet. (NAS, 1996) These figures do not take into account the ingestion of more contaminated food products, other sources of exposure, possible interactions of PCDDs and PCDFs with other chemicals, or increased incidence of cancer from the cancer promoting actions of dioxins. In addition, we reviewed average U.S. general population blood TEQ (ppt, lipid) levels, which reflect body burden, with levels of persons living in less industrialized countries. These data were used to compare the cancer risk of individuals living in the U.S., an industrial country, to those living in selected less industrialized countries. With the assumption that similar populations were being compared, it is estimated that in the non-industrial region of north Vietnam there is one-third the risk of dioxin-related cancer compared with the United States, while Cambodians have a dioxin-related cancer risk that is 14 fold less than the U.S. at this time.

Validation of precision-cut liver slices in dynamic organ culture as an in vitro model for studying CYP1A1 and CYP1A2 induction.

The utilization of precision-cut liver slices in dynamic organ culture as an in vitro model was validated by comparing the induction of the biomarker responses following in vitro (rat liver slice) and in vivo exposure of rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The biomarker responses investigated were cytochrome P450s 1A1 and 1A2 (CYP1A1 and CYP1A2) mRNA, protein, and activities. Precision-cut rat liver slices were incubated in dynamic organ culture for 24 hr with medium containing 0.001-10 nM TCDD or medium without TCDD (control). The resultant mean TCDD concentration in the slices ranged from 19 to 80,925 ppt (wet wt), respectively. A concentration-dependent induction of CYP1A1 mRNA, protein, and activities and a more modest induction of CYP1A2 mRNA was observed in liver slices at all medium concentrations of TCDD. The O-demethylation of 7-methoxyresorufin, a marker for CYP1A2 activity, was induced at TCDD medium levels of 0.01 nM and greater, whereas a detectable increase in CYP1A2 protein occurred only at the higher concentrations. Comparable liver concentrations of TCDD (8-64,698 ppt wet wt) were achieved at 24 hr following a single in vivo exposure of rats to TCDD at doses ranging from 0.002 to 5 microg/kg po. Concentration-effect and dose-response relationships for induction of CYP1A1 and CYP1A2 were similar following in vitro and in vivo exposure to TCDD, although the magnitude of induction was greater for in vivo exposure. The data support the use of liver slices in dynamic organ culture for assessing the relative in vivo potency of a compound to induce CYP1A1 and CYP1A2. Human tissue can also be readily utilized in this in vitro model to predict the biological and toxicological effects of a given in vivo exposure to TCDD.

Decrease in milk and blood dioxin levels over two years in a mother nursing twins: estimates of decreased maternal and increased infant dioxin body burden from nursing.

This study addresses the issue of breast-feeding and its reduction of maternal dioxin body burden. Nursing is also a source of infant dioxin exposure. This study extends our previous efforts to investigate a nursing mother's milk and blood dioxin levels. We report polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated dibenzofuran (PCDF) dioxin toxic equivalents (TEQs) in milk (M) and blood (B) both before and also after two years of nursing twins to be 16.9 ppt (M), 14.9 ppt (B), and 3.1 ppt (M) and 4.9 ppt (B), respectively. The ratios of measured congeners comparing milk to whole blood from a nursing mother taken initially and after two years of nursing vary from 0.36 to 8.40 in 1992 and 0.17 to 1.0 in 1994. The mother's body burden was initially calculated to be 329 ng TEQ from milk levels and 291 ng TEQ from blood levels using samples taken in February 1992 and decreased to 60.1 ng TEQ from milk and 96 ng TEQ from measured blood using samples collected in December 1994. We calculate that the excretion of dioxin TEQ by the mother through breast-feeding is 269 ng TEQ, which is similar to the 303 ng TEQ estimated total dioxin intake by the twins over two years. The average daily dioxin intake from nursing is 66 pg TEQ/kg-BW/day for each twin over the two years.

Calibrated 0.1-cm(-1) IR emission spectra from 80°N.

Spectra from a 0.1-cm(-1) resolution absolutely calibrated emission interferometer installed near Eureka, Northwest Territories, Canada (80°N, 86°W), at the Arctic Stratospheric Observatory are presented. The Michelson-type interferometer has a maximum path difference of 10 cm and uses a liquid-N(2)-cooled HgCdTe detector, which covers the spectral region from 650 to 1250 cm(-1). Spectral intervals containing CO(2), HNO(3), and ozone have been modeled with a line-by-line radiative-transfer code and column amounts retrieved for the latter two constituents. The instrument and initial measurements are described.

2,3,7,8-Tetrachlorodibenzo-p-dioxin induces diverse retinoic acid metabolites in multiple tissues of the Sprague-Dawley rat.

Retinoic acid metabolism was examined in microsomes prepared from four retinoid target tissues of male Sprague-Dawley rats removed 3 days after a single exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, 5 or 80 micrograms/kg, ip). Microsomes from all four tissues catalyzed increased rates of retinoic acid metabolism, with the degree of induction following the order: liver > lung = kidney = testis. The responses were tissue-specific with respect to the metabolites affected, the effects of dose, and the substrate used, [3H]retinoic acid or [3H]retinoic acid bound with cellular retinoic acid-binding protein. For example, neither 4-hydroxy- nor 18-hydroxy-retinoic acid increased in testis; 4-hydroxy- but not 18-hydroxy-retinoic acid increased in liver; and both 4-hydroxy- and 18-hydroxy-retinoic acid increased in kidney and lung. This ability of TCDD to affect diverse retinoic acid metabolites in multiple tissues, including those from a physiologically relevant substrate, holocellular retinoic-acid binding protein, strengthens the possibility that one aspect of TCDD toxicity involves altering the metabolism of retinoic acid.

Congener-specific levels of dioxins and dibenzofurans in U.S. food and estimated daily dioxin toxic equivalent intake.

Food, especially meat, milk, and fish, is the immediate source of almost all polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and dioxinlike compounds in the general population. To estimate intake of these highly toxic compounds, we performed congener-specific dioxin analyses for the first time on U.S. food for 18 dairy meat, and fish samples from a supermarket in upstate New York. 2,3,7,8 Tetrachlorodibenzo-p-dioxin (TCDD, "dioxin") toxic equivalents (TEqs) on a wet weight basis for the dairy products ranged for 0.04 to 0.7 ppt, meat TEqs ranged from 0.03 to 1.5 ppt, and fish TEqs ranged from 0.02 to 0.13 ppt. Previous human breast milk and infant formula analyses were used with the current preliminary food data to estimate a range of dioxin intake for Americans. Average daily food intake of TEqs for an adult weighing 65 kg was estimated to be between 0.3 and 3.0 pg/kg body weight, for a total of 18-192 pg TEq, using 1986 American consumption rates. Due to the relatively high level of PCDDs and PCDFs commonly found in human breast milk from American women and from women in other industrial countries, a nursing infant may consume an average of 35-53 pg TEq/kg body weight/day in its first year of life. This may be compared with the current U.S. EPA virtually safe dose of 0.006 pg TCDD/kg body weight per day over a 70-year lifetime based on an upper limit cancer risk of 10(-6), or the 10 pg/kg/day used by some European government agencies.

Hepatic uptake and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin and 2,3,7,8-tetrachlorodibenzofuran.

The pharmacokinetics of TCDD and related compounds is congener, dose, and species specific, with urinary and biliary excretion being dependent on the metabolism of these compounds. Isolated hepatocytes and liver slices in suspension culture and hepatic microsomes were used as in vitro models to assess the hepatic uptake and metabolism of [3H]- and [14C]TCDD and [3H]TCDF (0.01-1.0 microM) in control and induced (5 micrograms TCDD/kg, 3 days earlier) male Sprague-Dawley rats. TCDD pretreatment, with an increase in cytochromes P450 1A1 and 1A2 (CYP1A1, CYP1A2), produced an increase in the hepatic uptake of TCDD, while no increase in the hepatic uptake of TCDF was observed. The results are consistent with CYP1A2 serving as a hepatic binding protein for TCDD but not for TCDF. The rates of metabolism of TCDD and TCDF were directly proportional to their concentrations, indicating that the reaction follows first order kinetics at concentrations from 0.01 to 1.0 microM. Very limited metabolism of TCDD and TCDF was observed in control rat liver (0.45 and 3.2 pmol/hr/g hepatocyte wet wt at 0.1 microM, respectively). TCDD induced its own rate of metabolism about two- to fivefold at 1.0 microM but no induction was observed at 0.01 and 0.1 microM. In contrast, TCDD markedly induced the rate of TCDF metabolism at all substrate concentrations. While the results support the role of rat CYP1A1 in TCDF metabolism, the data suggest that CYP1A1 or CYP1A2 may not metabolize TCDD. These results also support the hypothesis that the more rapid metabolism and excretion of TCDF accounts for the relative resistance of the rat to the acute toxicity of TCDF. Comparative studies in rat and human liver microsomes found that TCDF metabolism exhibited first order kinetics in both species. Furthermore, the rate of TCDF metabolism in human liver microsomes was similar to that of control rat liver microsomes. Together the results suggest that TCDF will be far more persistent in rats, and possibly humans, following exposure at low doses which do not significantly induce cytochrome P450 1A1 and/or 1A2.