Prenatal Pb exposure is associated with reduced abundance of beneficial gut microbial cliques in late childhood: an investigation using Microbial Co-occurrence Analysis (MiCA).

Background: Many analytical methods used in gut microbiome research focus on either single bacterial taxa or the whole microbiome, ignoring multi-bacteria relationships (microbial cliques). We present a novel analytical approach to identify multiple bacterial taxa within the gut microbiome of children at 9-11 years associated with prenatal Pb exposure. Methods: Data came from a subset of participants (n=123) in the Programming Research in Obesity, Growth, Environment and Social Stressors (PROGRESS) cohort. Pb concentrations were measured in maternal whole blood from the second and third trimesters of pregnancy. Stool samples collected at 9-11 years old underwent metagenomic sequencing to assess the gut microbiome. Using a novel analytical approach, Microbial Co-occurrence Analysis (MiCA), we paired a machine-learning algorithm with randomization-based inference to first identify microbial cliques that were predictive of prenatal Pb exposure and then estimate the association between prenatal Pb exposure and microbial clique abundance. Results: With second-trimester Pb exposure, we identified a 2-taxa microbial clique that included Bifidobacterium adolescentis and Ruminococcus callidus, and a 3-taxa clique that added Prevotella clara. Increasing second-trimester Pb exposure was associated with significantly increased odds of having the 2-taxa microbial clique below the 50th percentile relative abundance (OR=1.03,95%CI[1.01-1.05]). In an analysis of Pb concentration at or above vs. below the United States and Mexico guidelines for child Pb exposure, odds of the 2-taxa clique in low abundance were 3.36(95%CI[1.32-8.51]) and 6.11(95%CI[1.87-19.93]), respectively. Trends were similar with the 3-taxa clique but not statistically significant. Discussion: Using a novel combination of machine-learning and causal-inference, MiCA identified a significant association between second-trimester Pb exposure and reduced abundance of a probiotic microbial clique within the gut microbiome in late childhood. Pb exposure levels at the guidelines for child Pb poisoning in the United States, and Mexico are not sufficient to protect against the potential loss of probiotic benefits.

Early-life metal exposure and schizophrenia: A proof-of-concept study using novel tooth-matrix biomarkers.

BACKGROUND: Despite evidence for the effects of metals on neurodevelopment, the long-term effects on mental health remain unclear due to methodological limitations. Our objective was to determine the feasibility of studying metal exposure during critical neurodevelopmental periods and to explore the association between early-life metal exposure and adult schizophrenia. METHODS: We analyzed childhood-shed teeth from nine individuals with schizophrenia and five healthy controls. We investigated the association between exposure to lead (Pb(2+)), manganese (Mn(2+)), cadmium (Cd(2+)), copper (Cu(2+)), magnesium (Mg(2+)), and zinc (Zn(2+)), and schizophrenia, psychotic experiences, and intelligence quotient (IQ). We reconstructed the dose and timing of early-life metal exposures using laser ablation inductively coupled plasma mass spectrometry. RESULTS: We found higher early-life Pb(2+) exposure among patients with schizophrenia than controls. The differences in log Mn(2+) and log Cu(2+) changed relatively linearly over time to postnatal negative values. There was a positive correlation between early-life Pb(2+) levels and psychotic experiences in adulthood. Moreover, we found a negative correlation between Pb(2+) levels and adult IQ. CONCLUSIONS: In our proof-of-concept study, using tooth-matrix biomarker that provides direct measurement of exposure in the fetus and newborn, we provide support for the role of metal exposure during critical neurodevelopmental periods in psychosis.

Relative potencies for acute effects of pyrethroids on motor function in rats.

The prevalence of pyrethroids in insecticide formulations has increased in the last decade. A common mode-of-action has been proposed for pyrethroids based on in vitro studies, which includes alterations in sodium channel dynamics in nervous system tissues, consequent disturbance of membrane polarization, and abnormal discharge in targeted neurons. The objective of this work was to characterize individual dose-response curves for in vivo motor function and calculate relative potencies for eleven commonly used pyrethroids. Acute oral dose-response functions were determined in adult male Long Evans rats for five Type I (bifenthrin, S-bioallethrin, permethrin, resmethrin, tefluthrin), five Type II (beta-cyfluthrin, lambda-cyhalothrin, cypermethrin, deltamethrin, esfenvalerate) and one mixed Type I/II (fenpropathrin) pyrethroids (n = 8-18 per dose; 6-11 dose levels per chemical, vehicle = corn oil, at 1 ml/kg). Motor function was measured using figure-8 mazes. Animals were tested for 1 h during the period of peak effects. All pyrethroids, regardless of structural class, produced dose-dependent decreases in motor activity. Relative potencies were calculated based on the computed ED30s. Deltamethrin, with an ED30 of 2.51 mg/kg, was chosen as the index chemical. Relative potency ratios ranged from 0.009 (resmethrin) to 2.092 (esfenvalerate). Additional work with environmentally-based mixtures is needed to test the hypothesis of dose-additivity of pyrethroids.

Protocols for the assurance of microarray data quality and process control.

Microarrays represent a powerful technology that provides the ability to simultaneously measure the expression of thousands of genes. However, it is a multi-step process with numerous potential sources of variation that can compromise data analysis and interpretation if left uncontrolled, necessitating the development of quality control protocols to ensure assay consistency and high-quality data. In response to emerging standards, such as the minimum information about a microarray experiment standard, tools are required to ascertain the quality and reproducibility of results within and across studies. To this end, an intralaboratory quality control protocol for two color, spotted microarrays was developed using cDNA microarrays from in vivo and in vitro dose-response and time-course studies. The protocol combines: (i) diagnostic plots monitoring the degree of feature saturation, global feature and background intensities, and feature misalignments with (ii) plots monitoring the intensity distributions within arrays with (iii) a support vector machine (SVM) model. The protocol is applicable to any laboratory with sufficient datasets to establish historical high- and low-quality data.

A unifying concept for assessing toxicological interactions: changes in slope.

Robust statistical methods are important to the evaluation of toxicological interactions (i.e., departures from additivity) among chemicals in a mixture. However, different concepts of joint toxic action as applied to the statistical analysis of chemical mixture toxicology data or as used in environmental risk assessment often appear to conflict with one another. A unifying approach for application of statistical methodology in chemical mixture toxicology research is based on consideration of change(s) in slope. If the slope of the dose-response curve of one chemical does not change in the presence of other chemicals, then there is no interaction between the first chemical and the others. Conversely, if the rate of change in the response with respect to dose of the first chemical changes in the presence of the other chemicals, then an interaction is said to exist. This concept of zero interaction is equivalent to the usual approach taken in additivity models in the statistical literature. In these additivity models, the rate of change in the response as a function of the i(th) chemical does not change in the presence of other chemicals in a mixture. It is important to note that Berenbaum's (1985, J. Theor. Biol. 114, 413-431) general and fundamental definition of additivity does not require the chemicals in the mixture to have a common toxic mode of action nor to have similarly shaped dose response curves. We show an algebraic equivalence between these statistical additivity models and the definition of additivity given by Berenbaum.

Neurotoxicological and statistical analyses of a mixture of five organophosphorus pesticides using a ray design.

Environmental exposures generally involve chemical mixtures instead of single chemicals. Statistical models such as the fixed-ratio ray design, wherein the mixing ratio (proportions) of the chemicals is fixed across increasing mixture doses, allows for the detection and characterization of interactions among the chemicals. In this study, we tested for interaction(s) in a mixture of five organophosphorus (OP) pesticides (chlorpyrifos, diazinon, dimethoate, acephate, and malathion). The ratio of the five pesticides (full ray) reflected the relative dietary exposure estimates of the general population as projected by the US EPA Dietary Exposure Evaluation Model (DEEM). A second mixture was tested using the same dose levels of all pesticides, but excluding malathion (reduced ray). The experimental approach first required characterization of dose-response curves for the individual OPs to build a dose-additivity model. A series of behavioral measures were evaluated in adult male Long-Evans rats at the time of peak effect following a single oral dose, and then tissues were collected for measurement of cholinesterase (ChE) activity. Neurochemical (blood and brain cholinesterase [ChE] activity) and behavioral (motor activity, gait score, tail-pinch response score) endpoints were evaluated statistically for evidence of additivity. The additivity model constructed from the single chemical data was used to predict the effects of the pesticide mixture along the full ray (10-450 mg/kg) and the reduced ray (1.75-78.8 mg/kg). The experimental mixture data were also modeled and statistically compared to the additivity models. Analysis of the 5-OP mixture (the full ray) revealed significant deviation from additivity for all endpoints except tail-pinch response. Greater-than-additive responses (synergism) were observed at the lower doses of the 5-OP mixture, which contained non-effective dose levels of each of the components. The predicted effective doses (ED20, ED50) were about half that predicted by additivity, and for brain ChE and motor activity, there was a threshold shift in the dose-response curves. For the brain ChE and motor activity, there was no difference between the full (5-OP mixture) and reduced (4-OP mixture) rays, indicating that malathion did not influence the non-additivity. While the reduced ray for blood ChE showed greater deviation from additivity without malathion in the mixture, the non-additivity observed for the gait score was reversed when malathion was removed. Thus, greater-than-additive interactions were detected for both the full and reduced ray mixtures, and the role of malathion in the interactions varied depending on the endpoint. In all cases, the deviations from additivity occurred at the lower end of the dose-response curves.

Normalization of two-channel microarray experiments: a semiparametric approach.

MOTIVATION: An important underlying assumption of any experiment is that the experimental subjects are similar across levels of the treatment variable, so that changes in the response variable can be attributed to exposure to the treatment under study. This assumption is often not valid in the analysis of a microarray experiment due to systematic biases in the measured expression levels related to experimental factors such as spot location (often referred to as a print-tip effect), arrays, dyes, and various interactions of these effects. Thus, normalization is a critical initial step in the analysis of a microarray experiment, where the objective is to balance the individual signal intensity levels across the experimental factors, while maintaining the effect due to the treatment under investigation. RESULTS: Various normalization strategies have been developed including log-median centering, analysis of variance modeling, and local regression smoothing methods for removing linear and/or intensity-dependent systematic effects in two-channel microarray experiments. We describe a method that incorporates many of these into a single strategy, referred to as two-channel fastlo, and is derived from a normalization procedure that was developed for single-channel arrays. The proposed normalization procedure is applied to a two-channel dose-response experiment.

Empirical bayes gene screening tool for time-course or dose-response microarray data.

An efficient method to reduce the dimensionality of microarray gene expression data from thousands or tens of thousands of cDNA clones down to a subset of the most differentially expressed cDNA clones is essential in order to simplify the massive amount of data generated from microarray experiments. An extension to the methods of Efron et al. [Efron, B., Tibshirani, R., Storey, J., Tusher, V. (2001). Empirical Bayes analysis of a microarray experiment. J. Am. Statist. Assoc. 96:1151-1160] is applied to a differential time-course experiment to determine a subset of cDNAs that have the largest probability of being differentially expressed with respect to treatment conditions across a set of unequally spaced time points. The proposed extension, which is advocated to be a screening tool, allows for inference across a continuous variable in addition to incorporating a more complex experimental design and allowing for multiple design replications. With the current data the focus is on a time-course experiment; however, the proposed methods can easily be implemented on a dose-response experiment, or any other microarray experiment that contains a continuous variable of interest. The proposed empirical Bayes gene-screening tool is compared with the Efron et al. (2001) method in addition to an adjusted model-based t-value using a time-course data set where the toxicological effect of a specific mixture of chemicals is being studied.

Temporal- and dose-dependent hepatic gene expression changes in immature ovariectomized mice following exposure to ethynyl estradiol.

Temporal- and dose-dependent changes in hepatic gene expression were examined in immature ovariectomized C57BL/6 mice gavaged with ethynyl estradiol (EE), an orally active estrogen. For temporal analysis, mice were gavaged every 24 h for 3 days with 100 microg/kg EE or vehicle and liver samples were collected at 2, 4, 8, 12, 24 and 72 h. Gene expression was monitored using custom cDNA microarrays containing 3067 genes/ESTs of which 393 exhibited a change at one or more time points. Functional gene annotation extracted from public databases associated temporal gene expression changes with growth and proliferation, cytoskeletal and extracellular matrix responses, microtubule-based processes, oxidative metabolism and stress, and lipid metabolism and transport. In the dose-response study, hepatic samples were collected 24 h following treatment with 0, 0.1, 1, 10, 100 or 250 microg/kg EE. Thirty-nine of the 79 genes identified as differentially regulated at 24 h in the time course study exhibited a dose-response relationship with an average ED50 value of 47 +/- 3.5 microg/kg. Comparative analysis indicated that many of the identified temporal and dose-dependent hepatic responses are similar to EE-induced uterine responses reported in the literature and in a companion study using the same animals. Results from these studies confirm that the liver is a highly estrogen responsive tissue that exhibits a number of common responses shared with the uterus as well as distinct estrogen-mediated profiles. These data will further aid in the elucidation of the mechanisms of action of estrogens in the liver as well as in other classical and non-classical estrogen responsive tissues.

Autoregulation of cerebral blood flow in fetuses with congenital heart disease: the brain sparing effect.

Fetuses with congenital heart disease (CHD) have circulatory abnormalities that may compromise cerebral oxygen delivery. We believe that some CHD fetuses with decreased cerebral oxygen supply have autoregulation of blood flow that enhances cerebral perfusion (brain sparing). We hypothesize that cerebral autoregulation occurs in CHD fetuses, and the degree of autoregulation is dependent on the specific CHD and correlates with intrauterine head circumferences. CHD fetuses were compared to normal fetuses. Data included cardiac diagnosis, cerebral and umbilical artery Doppler, head circumference, weight, and gestational age. The cerebral-to-placental resistance ratio (CPR) was assessed as a measure of cerebral autoregulation. CPR = cerebral/umbilical resistance index (RI) and RI = systolic-diastolic/systolic velocity (normal CPR > 1). CPR > 1 was found in 95% of normal vs 44% of CHD fetuses. The incidence of CPR < 1 was greatest in hypoplastic left or right heart fetuses. Compared to normal, cerebral RI was decreased in CHD fetuses. The CPR vs gestational age relationship, and the relationship among weight, head circumference, and CPR differed across normal and CHD fetuses. Fetuses > 2 kg with CHD and a CPR < 1 had smaller head circumferences than normal. Brain sparing occurs in CHD fetuses. Fetuses with single ventricular physiology are most affected. Inadequate cerebral flow in CHD fetuses, despite autoregulation, may alter brain growth.

Identification of temporal patterns of gene expression in the uteri of immature, ovariectomized mice following exposure to ethynylestradiol.

Estrogen induction of uterine wet weight provides an excellent model to investigate relationships between changes in global gene expression and well-characterized physiological responses. In this study, time course microarray GeneChip data were analyzed using a novel approach to identify temporal changes in uterine gene expression following treatment of immature ovariectomized C57BL/6 mice with 0.1 mg/kg 17alpha-ethynylestradiol. Functional gene annotation information from public databases facilitated the association of changes in gene expression with physiological outcomes, which allowed detailed mechanistic inferences to be drawn regarding cell cycle control and proliferation, transcription and translation, structural tissue remodeling, and immunologic responses. These systematic approaches confirm previously established responses, identify novel estrogen-regulated transcriptional effects, and disclose the coordinated activation of multiple modes of action that support the uterotrophic response elicited by estrogen. In particular, it was possible to elucidate the physiological significance of the dramatic induction of arginase, a classic estrogenic response, by elucidating its mechanistic relevance and delineating the role of arginine and ornithine utilization in the estrogen-stimulated induction of uterine wet weight.

Neurobehavioral evaluations of mixtures of trichloroethylene, heptachlor, and di(2-ethylhexyl)phthalate in a full-factorial design.

One approach to the toxicological evaluation of chemical mixtures is to construct full dose-response curves for each compound in the presence of a range of doses of each of the other compounds, i.e., a factorial design. This study was undertaken as part of an interdisciplinary project to evaluate a mixture of three environmental pollutants. A full-factorial design was undertaken to determine the neurobehavioral consequences of short-term repeated exposure to five dose levels each of three chemicals, in order to characterize potential two- and three-way interactions. Adult female F344 rats received (p.o.) for 10 days either one of five doses of trichloroethylene, di(2-ethylhexyl)phthalate, or heptachlor, or else one of all possible chemical combinations. Neurobehavioral evaluations were conducted using motor activity and an abbreviated functional observational battery. Response-surface analysis was applied to each of the endpoints. Hypotheses were tested based on the estimated model parameters; of primary interest was the overall test for interaction among the three chemicals. In addition, an abbreviated design was created by fitting only a subset of the data to the model. In general, significant overall interactions that deviated from response additivity were detected for most endpoints (11 of 14). All of the interactions on the neurobehavioral endpoints showed either antagonism, or else an interaction that could not be fully characterized. Often the results of the abbreviated dataset analysis were not the same as for the full-factorial design. This study was extremely intensive, in terms of the number of rats and time required for conduct of the study as well as the data analysis. These results underscore the need for more economical approaches to evaluate the toxic effects of mixtures of chemicals.

An approach for assessing estrogen receptor-mediated interactions in mixtures of three chemicals: a pilot study.

Most studies investigating interactions among endocrine-active chemicals have been limited to binary mixtures. This study reports on the preliminary evaluation an in vitro MCF-7 cell ER-alpha reporter gene system, coupled with a statistical methodology adapted for assessing interactions within ternary (3-chemical) mixtures. Two mixtures were initially chosen for assessment of the in vitro system's ability to detect additivity (mixture A) as well as greater-than-additive (mixture B) responses. Mixture A was composed of 17beta-estradiol (E2), ethinyl estradiol, and diethylstilbestrol and served as a control for additivity, whereas mixture B (E2, epidermal growth factor, insulin-like growth factor-I) was selected to model greater-than-additive interactions based on previous in vitro studies. After generating complete dose-response curves for each chemical, ternary mixtures were then tested in a full factorial design (4 concentrations per chemical, 64 treatment groups). A response surface was estimated using a nonlinear mixed model, and the observed responses were statistically analyzed for departures from the responses expected under the assumption of additivity. Mixture A exhibited additivity in vitro when the chemicals were present at concentrations in the linear range of their individual dose-response curves. For mixture B, in vitro analysis resulted in the additivity hypothesis being rejected (p < 0.001) because of a greater-than-additive interaction, as expected. A limited in vivo evaluation of mixture A was performed in the immature mouse uterotrophic assay (27 treatment groups), which agreed with the in vitro assessment of no significant departure from additivity ( p = 0.903). These findings demonstrate the ability of this in vitro methodology to detect additive, greater-than-additive, and less-than-additive interactions within ternary mixtures, which now allows for the assessment of environmentally relevant mixtures.

Assessment of interactions of diverse ternary mixtures in an estrogen receptor-alpha reporter assay.

This study used an MCF-7 cell based ER-alpha reporter gene assay to assess chemical interactions within the following ternary mixtures: (1) three synthetic pesticides, methoxychlor (MXC), o,p-DDT, and dieldrin; (2) three polyaromatic hydrocarbons, benzo[a]pyrene (BAP), 1,2-benzanthracene (BENZ), and chrysene (CHRY); and (3) an endogenous estrogen, [17beta-estradiol, (E(2))]; a phytoestrogen, genistein (GEN); and a synthetic estrogen, o,p-DDT. A full factorial design in which four concentrations of each chemical were assessed in all possible combinations (64 treatment groups) was utilized. In addition, mixtures were tested in both a low range (concentrations near the individual chemical response thresholds) and a high range ( approximately 2-10x higher) experiment. A response surface was estimated using a nonlinear mixed model, and the cumulative response in each mixture was evaluated for departure from additivity. The mixture of E(2), GEN, and DDT exhibited antagonistic interactions (p < 0.001) in both concentration ranges. However, specific interactions between E(2)/GEN and E(2)/DDT differed between the low and high range concentrations. The BAP/BENZ/CHRY mixture did not depart significantly from additivity (p = 0.66) in either concentration range, although response levels were generally low. The MXC/DDT/dieldrin mixture did not depart significantly from additivity in either the high (p = 0.065), or low dose range (p = 0.506), with generally minimal responses dominated by MXC and DDT. This methodology has allowed for a rigorous statistical evaluation of potential departures from additive interactions in endocrine active mixtures. In no case was a significantly greater-than-additive (synergistic) interaction observed.

Toxicological interactions among arsenic, cadmium, chromium, and lead in human keratinocytes.

To evaluate health effects of chemical mixtures, such as multiple heavy metals in drinking water, we have been developing efficient and accurate hazard identification strategies. Thus, in this study, we determine the cytotoxicity of arsenic, cadmium, chromium, and lead, and characterize interactions among these metals in human epidermal keratinocytes. Three immortal keratinocyte cell lines (RHEK-1, HaCaT, and NM1) and primary keratinocytes (NHEK) were used. A statistical approach applying an additivity response surface methodology was used to test the validity of the additivity concept for a 4-metal mixture. Responses of the 4 keratinocyte strains to the metal mixture were highly dose-dependent. A growth stimulatory effect (hormesis) was observed in RHEK-1, NM1, and NHEK cells with the metal mixture at low concentrations (low ppb range). This hormesis effect was not significant in HaCaT. As the mixture concentration increased, a trend of additivity changed to synergistic cytotoxicity in all 4 cell strains. However, in NHEK, RHEK-1, and HaCaT, at the highest mixture concentrations tested, the responses to the metal mixtures were antagonistic. In NM1, no significant antagonistic interaction among the metals was observed. To explore a mechanistic basis for these differential sensitivities, levels of glutathione and metallothioneins I and II were determined in the keratinocyte cell strains. Initial data are consistent with the suggestion that synergistic cytotoxicity turned to antagonistic effects because at highest mixture exposure concentrations cellular defense mechanisms were enhanced.

The vitamin D3 analog, ILX-23-7553, enhances the response to adriamycin and irradiation in MCF-7 breast tumor cells.

UNLABELLED: Ionizing radiation and the anthracycline antibiotic, Adriamycin, generally fail to promote a primary apoptotic response in experimental breast tumor cell lines. Similarly, the primary response of breast tumor cells to vitamin D3 (1,25(OH)2D3) and vitamin D3 analogs such as EB 1089 is growth inhibition. Previous studies have demonstrated that pretreatment of MCF-7 breast tumor cells with vitamin D3 or EB 1089 can increase sensitivity to both Adriamycin and irradiation. PURPOSE: The capacity of the vitamin D3 analog, ILX 23-7553, to enhance the antiproliferative and cytotoxic effects of Adriamycin or irradiation and to promote apoptosis in MCF-7 breast tumor cells was assessed in the present study. RESULTS: Pretreatment of MCF-7 cells with ILX 23-7553 followed by Adriamycin or irradiation decreased viable cell numbers by 97% and 93%, respectively. Cell numbers were reduced by 56%, 74% and 75% by ILX 23-7553, Adriamycin and irradiation alone. Pretreatment with ILX 23-7553 also shifted the dose response curve for clonogenic survival, increasing sensitivity to Adriamycin 2.5-fold and sensitivity to radiation fourfold. In addition, ILX 23-7553 pretreatment conferred sensitivity to Adriamycin- or irradiation-induced DNA fragmentation and resulted in morphological changes indicative of apoptotic cell death in MCF-7 cells. Statistical analysis demonstrated that ILX 23-7553 interacts additively and not synergistically with both Adriamycin and irradiation. CONCLUSIONS: ILX 23-7553 enhances the effects of Adriamycin and irradiation in MCF-7 breast tumor cells by decreasing viable cell numbers, reducing clonogenic survival and inducing apoptotic cell death. Current studies are focused on elucidating the mechanisms underlying the induction of apoptosis as well as understanding the nature of the interactions between ILX 23-7553 and Adriamycin or irradiation.

Antifungal antibiotics and breakthrough bacteremias.

Patients with fever and neutropenia are at high risk for infection ( approximately 50%) and bacteremia ( approximately 20%). As a result, most are treated with antibacterial prophylaxis until their absolute neutrophil count exceeds 500 cells/mm(3) and their temperature returns to normal. The 1997 guidelines of the Infectious Diseases Society of America suggested 1 of 3 regimens: vancomycin plus ceftazidime, monotherapy with ceftazidime or imipenem (possibly cefepime or meropenem), or dual therapy with an aminoglycoside plus an antipseudomonal beta-lactam.

On testing for drug/chemical interactions: definitions and inference.

The notion of zero interaction in the statistical literature is not always equivalent to what is found in the toxicology literature. A discussion about when they are the same is provided here. Design issues are of paramount importance in the analysis of drug combinations (mixtures of chemicals) when the number of constituents in the combination is larger than, say, three as the usual factorial designs are not feasible. An economical design necessary and sufficient to support the estimation of an additivity model is single drug (chemical) dose-response data. Once estimated, the additivity surface can be used to make comparisons to the observed data at combination points of interest. Examples are provided to demonstrate the methods.

Incorporation of S-9 activation into an ER-alpha transactivation assay.

We evaluated the feasibility of incorporating an exogenous metabolic activating system into an estrogen receptor-alpha transactivation assay. 17beta-estradiol (E2), and the proestrogenic pesticide methoxychlor (MXC) were evaluated for activity in the presence and absence of Aroclor-1254 induced rat liver S-9 fractions. Both E2 and MXC responded consistently in the assay with average EC(50) values of 9.6 x 10(-11) M and 1.2 x 10(-5) M, respectively. In the presence of a 0.1% S-9 fraction, the EC(50) for E2 was increased to 1.4 x 10(-9) M and that for MXC decreased to 4.9 x 10(-7) M, with both compounds demonstrating increased secondary metabolite formation as evidenced by HPLC analysis. Consistent with these data, metabolites of E2 and MXC exhibited decreased and increased potencies, respectively, in the assay system relative to the parent molecules. S-9 was compatible with the MCF-7 reporter assay and has the potential to enhance detection of proestrogenic materials.

A multiple-purpose design approach to the evaluation of risks from mixtures of disinfection by-products.

Drinking water disinfection has effectively eliminated much of the morbidity and mortality associated with waterborne infectious diseases in the United States. Various disinfection processes, however, produce certain types and amounts of disinfection by-products (DBPs), including trihalomethanes (THM), haloacetic acids, haloacetonitriles, and bromate, among others. Human health risks from the ubiquitous exposure to complex mixtures of DBPs are of concern because existing epidemiologic and toxicologic studies suggest the existence of systemic or carcinogenic effects. Researchers from several organizations have developed a multiple-purpose design approach to this problem that combines efficient laboratory experimental designs with statistical models to provide data on critical research issues (e.g., estimation of human health risk from low-level DBP exposures, evaluation of additivity assumptions as useful for risk characterization, estimation of health risks from different drinking water treatment options). A series of THM experiments have been designed to study embryonic development, mortality and cancer in Japanese medaka (Oryzias latipes) and liver and kidney endpoints in female CD-1 mice. The studies are to provide dose-response data for specific mixtures of the 4 THMs, for the single chemicals, and for binary combinations. The dose-levels and mixing ratios for these experiments were selected to be useful for development and refinement of three different statistical methods: testing for departures from dose-additivity; development of an interactions-based hazard index; and use of proportional-response addition as a risk characterization method. Preliminary results suggest that dose-additivity is a reasonable risk assessment assumption for DBPs. The future of mixtures research will depend on such collaborative efforts that maximize the use of resources and focus on issues of high relevance to the risk assessment of human health.