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.

A predictive model that evaluates the effect of growth conditions on the thermal resistance of Listeria monocytogenes.

A predictive model for Listeria monocytogenes was developed using cells grown in different pH and milkfat levels before subsequent thermal inactivation in identical pH and milkfat conditions. Inactivation of the cells used combinations of temperature (55, 60, 65 degrees C), pH (5.0, 6.0, 7.0), and milkfat (0%, 2.5%, 5.0%) in a complete 3 x 3 x 3 factorial design with each test done in triplicate. A modified Gompertz equation was used to model nonlinear survival curves with the following three parameter estimates: A for the shouldering region, B for the maximum death rate, and C for the tailing region. All treatment sets were analyzed together in a regression model using the modified Gompertz equation. There was good confidence in the overall model when it was used to predict values for the entire data set. The correlation of determination, R2, between the observed log surviving fraction (LSF) of cells from each of the conditions studied in the experiment, for the overall model was 0.811. For the A and B parameter estimates, temperature or milkfat alone, and the interaction of temperature and milkfat significantly (p < 0.05) affected the shouldering region and maximum death rate of a survival curve, respectively. These results were compared to a previously published predictive model, generated for cells grown under optimum conditions (pH 7.0, 0% milkfat), where pH was the only significant (p < 0.05) factor affecting the shoulder region. These results suggested that the conditions of the growth environment had an important impact on survival curve shape and the estimates of the predictive model. Specifically, there were more factor interactions involving temperature and milkfat level. These growth factors affected the shoulder region and maximum rate of death of the survival curve when cells were grown in identical medium conditions to which they were heated. Differences related to shouldering and inactivation rates for cells grown in different conditions may have important and practical importance for estimating inactivation of L. monocytogenes. This study provides some evidence on the importance of growing conditions when evaluating microbial heat resistance.

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.

Obstructive nephropathy in children: long-term progression after relief of posterior urethral valve.

BACKGROUND: Approximately one third of children with end-stage renal disease have the illness because of urinary tract malformations, obstructive uropathy, and hypoplasia/dysplasia. The significant drop in infant mortality from obstructive uropathies in recent decades, attributable to prenatal diagnosis with renal ultrasonography and coordinated surgical and medical care, necessitated a reevaluation of the long-term outcome. METHODS: To that end, we examined the long-term progression of obstructive nephropathy after neonatal relief of posterior urethral valves in our center over a span of 21 years, with diagnosis and care being provided by the same pediatric and urology team. RESULTS: The 10 consecutive cases of posterior urethral valves represented 7% of all patients with congenital malformative uropathies seen over this period. The following procedures were performed: primary valve ablation (90%) and vesicostomy (40%). Seventy percent of patients progressed to end-stage renal disease over a (mean +/- standard error of the mean) follow-up of 11.3 +/- 2.1 years. The linear plot of the log of the inverse of serum creatinine versus time suggested unrelenting progression. The rate of progression was rapid after serum creatinine exceeded 5 mg/dL but the rate was slow and steady from serum creatinine of 1.5 to 5 mg/dL. CONCLUSIONS: To test the effect of a therapeutic intervention to ameliorate the rate of progression, this steady and prolonged progression of 0.5 mg/dL per year between serum creatinine concentration of 1.5 to 5 mg/dL would seem the optimal study.

17 alpha androstenediol inhibition of breast tumor cell proliferation in estrogen receptor-positive and -negative cell lines.

Androstene-3beta, 17alpha-diol (17alpha-AED) inhibits DNA synthesis and induces apoptosis in several myeloid cancer cell lines. The purpose of this study was to determine if 17alpha-AED inhibition of human breast carcinoma cell proliferation is dependent on the estrogen or androgen receptor. At concentrations of 12.5 to 50 x 10(-9) M 17alpha-AED inhibited the proliferation of ZR75-1, estrogen receptor-positive (ER+) cells, by 54% to 68%. Further, 17alpha-AED inhibited MDA-MB231, estrogen receptor-negative (ER-) cells, by 33.6% to 56.0%. The inhibitory effect was dose dependent with a minimal effective inhibitory dose at 12.5x10(-9) M for both cell lines. Both 17beta-AED and estradiol potentiate the inhibitory effect of 17alpha-AED on ER+ cells at lower doses (3.13 to 6.25 x 10(-9) M) where 17alpha-AED alone was not inhibitory. The inhibitory action of 17alpha-AED on human mammary carcinomas appears to be independent of either the alpha estrogen or the androgen receptors.

A predictive model to determine the effects of pH, milkfat, and temperature on thermal inactivation of Listeria monocytogenes.

Listeria monocytogenes is a foodborne pathogen of significance because of its comparatively high heat resistance, zero tolerance in ready-to-eat foods, and growth at refrigeration temperatures. A 3 x 3 x 3 factorial study was done to determine the effects of milkfat (0%, 2.5%, 5.0%), pH (5.0, 6.0, 7.0), and processing temperature (55 degrees C, 60 degrees C, 65 degrees C) on the thermal resistance of L. monocytogenes in a formulated and homogenized milk system. Data were fit to a modified Gompertz equation where parameter estimates characterized three regions of a survival curve: the shoulder, maximum slope, and tail. Statistical analysis was done for each of the 27 individual treatment sets to visualize individual effects on parameter estimates and to evaluate how well the Gompertz equation represented the data. A regression model for the Gompertz equation was generated to predict the logarithmic surviving fraction of L. monocytogenes based on all 27 treatments and their single and interactive effects. The shoulder region of the survival curve was affected by pH; however, the maximum slope was affected by temperature, milkfat, and the interaction of temperature x milkfat. Validation of the model suggests that the predictions are best suited for processing above 62 degrees C. Trends over time for a 4-log reduction in cells (4D values) were evaluated using results from the 27 individual treatment sets, the regression model for the Gompertz equation, and a linear equation. At lower temperatures, 4D values by the three methods varied by twofold. At higher temperatures, all methods gave similar 4D values, suggesting that death became more linear. Based on this study all three factors affect heat resistance for specific regions of a survival curve, and a predictive model was developed that can be used as a preliminary estimate for L. monocytogenes inactivation.

Lung tumorigenic interactions in strain A/J mice of five environmental polycyclic aromatic hydrocarbons.

The binary, ternary, quaternary, and quintary interactions of a five-component mixture of carcinogenic environmental polycyclic aromatic hydrocarbons (PAHs) using response surface analyses are described. Initially, lung tumor dose-response curves in strain A/J mice for each of the individual PAHs benzo[a]pyrene (B[a]P), benzo[b]fluoranthene (B[b]F), dibenz[a,h]anthracene (DBA), 5-methylchrysene (5MC), and cyclopenta[cd]pyrene (CPP) were obtained. From these data, doses were selected for the quintary mixture study based on toxicity, survival, range of response, and predicted tumor yields. The ratios of doses among PAHs were designed to simulate PAH ratios found in environmental air and combustion samples. Quintary mixtures of B[a]P, B[b]F, DBA, 5MC, and CPP were administered to male strain A/J mice in a 2(5) factorial 32-dose group dosing scheme (combinations of five PAHs each at either high or low doses) and lung adenomas were scored. Comparison of observed lung adenoma formation with that expected from additivity identified both greater than additive and less than additive interactions that were dose related i.e., greater than additive at lower doses and less than additive at higher doses. To identify specific interactions, a response surface analysis using response addition was applied to the tumor data. This response surface model contained five dose, ten binary, ten ternary, five quaternary, and one quintary parameter. This analysis produced statistically significant values of 16 parameters. The model and model parameters were evaluated by estimating the dose-response relationships for each of the five PAHs. The predicted dose-response curves for all five PAHs indicated a good estimation. The binary interaction functions were dominated for the most part by DBA and were inhibitory. The response surface model predicted, to a significant degree, the observed lung tumorigenic responses of the quintary mixtures. These data suggest that although interactions between PAHs do occur, they are limited in extent.

Underreading of the tuberculin skin test reaction.

STUDY OBJECTIVE: The tuberculin skin test is the best diagnostic method to detect tuberculous infection. How accurate is interpretation of the test? DESIGN: Observational study. SETTING: Both general hospital and university hospital. PARTICIPANTS: One hundred seven health-care professionals, including 52 practicing pediatricians, 33 pediatric house officers, 10 pediatric academicians, 11 registered nurses, and 1 pediatric nurse practitioner. STUDY: A tuberculin skin test (Mantoux) was applied to the arm of a known tuberculin converter. As participants entered/left the room, they were guided to the tuberculin converter. At no time did a participant observe readings other than his/her own. RESULTS: Mantoux tuberculin reaction measuring 15 mm induration was read individually by a group of 52 practicing pediatricians, 33 pediatric house officers, 10 pediatric academicians, 11 registered nurses, and one pediatric nurse practitioner. The median induration recorded by this group of 107 health-care professionals was 10 mm, and 17 (33%) practicing pediatricians read the reaction as <10 mm induration. Using the > or = 15-mm induration indicator to identify a positive reaction, 93% of those in the study (99/107 participants) would have identified our known converter as tuberculin negative. CONCLUSION: This study confirms a general inaccuracy in interpretation of the tuberculin skin test reaction. It raises two questions. (1) Is there a general tendency toward underreading? (2) Does this general tendency to underread tuberculin skin test reactions raise some question as to the American Academy of Pediatrics, American Thoracic Society, and Centers for Disease Control and Prevention move in raising the amount of induration considered tuberculin positive to 15 mm in low-risk individuals?

Two-stage designs for the logistic regression model in single-agent bioassays.

In this paper we focus on the use of a two-stage procedure for logistic regression that emphasizes predicting response through the use of the Q-optimality criterion. The use of D-optimality in the first stage is primarily to allow best possible parameter estimates as one enters the second stage. However, it is important to understand that there are many ways to formulate the two-stage procedure. It may involve any optimality criterion in either stage. In fact, theoretically, one need not stop at two stages. It was our intention in this paper to demonstrate the potential in the two-stage procedure in cases in which good initial parameter estimates are not available. Those investigators who are interested in the software for the two-stage procedure described here should contact Dr. William R. Myers.

Use of a Modified Gompertz Equation to Predict the Effects of Temperature, pH, and NaCl on the Inactivation of Listeria monocytogenes Scott A Heated in Infant Formula.

The heat resistance of Listeria monocytogenes was determined in infant formula for all possible combinations of temperature (50, 55, and 60°C), pH level (5, 6, and 7), and NaCl concentration (0, 2, and 4%). Survival curves were fit using nonlinear regression with a Gompertz equation. The Gompertz equation was flexible enough to fit the three most commonly observed survival curves: linear curves, those with an initial lag region followed by a linear region, and sigmoidal shaped. Parameter estimates obtained by the method of nonlinear least squares were used to describe the effect(s) of different heating treatments on the lag region, death rate, and tailing region of survival curves. These estimates were further used to predict single and interactive effects of temperature, pH, and percentage of NaCl on the log of the surviving fraction (LSF) of bacteria. Interactions among these variables significantly (P ≤ .05) affected the LSF. Generally, increased pH or NaCl concentration lead to an increased LSF, whereas increased time or temperature lead to a decreased LSF. All multiple-factor interactions significantly (P ≤ .05) affected the LSF. The correlation of observed LSF versus predicted LSF (R2 = .92) indicated that the estimated Gompertz equation was in close agreement with the observation. This study demonstrated that the Gompertz equation and nonlinear regression can be used as an effective means to predict survival curve shape and response to heat of L. monocytogenes under many different environmental conditions.

Relating isobolograms to response surfaces.

The interaction index, isobologram and the appropriate contour of constant response of a dose response surface each offer essentially equivalent information regarding departures from additivity in a chemical combination. The benefit of relating the interaction index and isobolograms to a contour of a fitted dose response surface is that departures from additivity can be related to parameters of a statistical model. This permits an assessment of the statistical significance of the parameters and conclusions regarding the nature of departures from additivity which account for the variability inherent in the experiments used to generate the data. Relationships between statistical models and experimental designs can be exploited to yield economical designs for studying chemical combinations.

Analysis of anticonvulsant and neurotoxic responses to combination therapy with carbamazepine, felbamate and phenytoin by response-surface modeling.

Various combinations of carbamazepine (CAS 298-46-4), felbamate (CAS 25451-15-4), and phenytoin (CAS 57-41-0) were evaluated in mice (i.p.) for anticonvulsant activity (maximal electroshock seizure test) and minimal neurotoxicity (rotarod test). The results obtained from these studies were analyzed using response surface methodologies (RSM). The outcomes of these analyses in regard to anticonvulsant activity suggest that, under these experimental study conditions, at 0.5 h post treatment there is a significant carbamazepine/phenytoin synergism even though none of the drugs has a significant dose-response by that time when given alone, and that at 1.0 h post treatment, the combination dose-response is additive. Thus, there appears to be an important dose/time relationship. In regard to the neurotoxic response, the results suggest a significant carbamazepine/phenytoin synergism at 0.25 h post treatment and an additive neurotoxic effect due to the combination of felbamate/carbamazepine/phenytoin at 0.5, 1.0 and 2.0 h post exposure.

Use of a Modified Gompertz Equation to Model Nonlinear Survival Curves for Listeria monocytogenes Scott A.

The heat resistance of Listeria monocytogenes was determined in 0.1 M KH2PO4 buffer at three temperatures (50, 55, and 60°C), three pH levels (5, 6, and 7), and three NaCl concentrations (0, 2, and 4%). Survival curves were fit using nonlinear regression with a modified Gompertz equation. The Gompertz equation is capable of fitting survival curves which are linear, those which display an initial lag region followed by a linear region, and those which are sigmoidal. Parameter estimates were used to describe the lag region, death rate, and the tailing region of a survival curve. These estimates were also used to predict single and interactive effects of temperature, pH, and percentage of NaCl on the log surviving fraction (LSF) of bacteria. Interactions among these variables significantly (P < .05) affected the LSF. Generally, increased pH or NaCl concentration lead to an increased (P < .05) LSF, where as increased time or temperature lead to a decreased (P < .05) LSF. All multiple factor interactions significantly (P < .05) affected the LSF. These interactions differed depending on the heating medium and the region of the survival curve. The correlation of observed LSF and predicted LSF (R2 = .89) indicated that the Gompertz equation was in close agreement with the observations. This study demonstrated that the Gompertz equation and nonlinear regression can be used as an effective means to predict survival curve shape and response to heat of L. monocytogenes in many different environmental conditions.

Efficacy comparison of scopolamine and diazepam against soman-induced debilitation in guinea pigs.

The efficacy of diazepam (DZ) and scopolamine (SCP), in combination with atropine (ATR)+oxime therapy, against soman-induced seizure/convulsive activity and associated brain damage has been demonstrated, but the efficacy of each against the incapacitating effects of soman has not been addressed. Thus, the therapeutic efficacies of SCP (5 doses; 0-0.86 mg/kg) and DZ (5 doses; 0-5 mg/kg), when each was used in conjunction with ATR (3 doses; 0.5-8 mg/kg) + 2-PAM (25 mg/kg) therapy, were compared in groups of pyridostigmine pretreated guinea pigs exposed to 1.6, 2.0, 2.5, or 3.2 LD50s of soman. Response surface methodology was employed to describe the relationship between soman-induced incapacitation and the ATR/DZ or ATR/SCP dosages. Incapacitation was measured by toxicity scores assigned by three graders to test animals at 60 min postsoman. Results show that as the dosage of SCP increased, the mean toxicity scores decreased. Also, within the indicated dose ranges used, the efficacy of SCP was not dependent on the presence of ATR. In contrast, ATR alone was found to be more effective than when combined with DZ at any dose, and indicates that DZ might be temporarily contributing to soman-induced incapacitation. These findings suggest that in guinea pigs, SCP could replace ATR or DZ, or both, as therapy against soman-induced incapacitation.

Treatment outcomes in patients with adult thrombotic thrombocytopenic purpura-hemolytic uremic syndrome.

BACKGROUND: Plasma treatment has improved the outcomes in adults with thrombotic thrombocytopenic purpura (TTP)-hemolytic uremic syndrome (HUS). We reviewed our experience in treating unselected patients to determine the clinical outcomes and to evaluate the treatments given in addition to plasma. METHODS: A chart review of all cases of TTP and HUS in adults treated at the Toronto (Ontario) Hospital, the largest treatment center for adults with TTP-HUS in the province of Ontario, was conducted. RESULTS: Sixty-seven episodes of TTP-HUS in 52 consecutive adult patients were treated during a 12-year period. Plasma was the primary form of therapy, and most patients received plasma exchange. A complete hematologic remission was achieved in 65 of 67 episodes; however, two patients in remission were brain-dead. The time to complete remission varied from 3 to 58 days (median, 13 days). The death rate during the acute illness was 8%. Long-term sequelae included relapses, persisting renal impairment, hepatitis, and transfusion-associated acquired immunodeficiency syndrome. Relapses occurred in 21% of patients during a median follow-up of 1.1 years (range, 0.1 to 18 years). Analyses of the treatment given in addition to plasma did not demonstrate a significant benefit in terms of reducing the illness duration, mortality, or long-term sequelae. CONCLUSION: While most patients recovered from TTP-HUS, deaths still occurred and many patients suffered long-term complications. The role of the treatments given in addition to plasma is uncertain.

Multiple drug chemotherapy combined with multiple fractions per day (MFD) radiotherapy in an experimental solid tumor model.

A series of multi-drug chemotherapy studies combined with multi fractions per day (MFD) radiotherapy have been carried out in an experimental solid tumor model to provide information on how drugs contribute in combinations, both for effect on tumor and with regard to host toxicity. Twenty-five different combinations of cyclophosphamide (CP) and 5-fluorouracil (FU) were evaluated in this initial study. All tumors received a total dose of 6000 cGy of radiation given in multiple fractions per day for three courses one week after combined chemotherapy. Doses of each drug in the combinations ranged from 25 to 100% of the LD10 (150 mg/kg or .9 mg/m2) of each drug when given as a single agent. Proportional hazards analysis of the survival data yielded optimal doses of 132.8 mg/kg FU and 115.0 mg/kg CP, that is, approximately 3/4 of the LD10 of each drug in the combination. The group close to the optimum combination had a median GD of 295.3 days. Among the 5 rats in this group there were 4 partial responses and 1 cure for a 100% response rate. Excessive toxicity occurred in the group given the highest doses of FU and CP along with the 6000 cGy radiotherapy. These experimental cancer treatment studies in well defined solid tumor models demonstrate how the quantitative inter-relationship between anti-tumor effects and host toxicity to tumor burden and total therapeutic dose chemotherapy (single or multiple combinations) and radiotherapy alone or in combination can be obtained. These major determinants on treatment outcome are difficult or impossible to determine in clinical studies.(ABSTRACT TRUNCATED AT 250 WORDS)