Russell E. Savage

Comparative metabolism of bis(2-methoxyethyl)ether in isolated rat hepatocytes and in the intact rat: effects of ethanol on in vitro metabolism.

The metabolism of the reproductive and developmental toxicant bis(2-methoxyethyl)ether (diglyme) was studied in isolated rat hepatocytes and in the intact rat. Male Sprague-Dawley rats (190–220 g) were used in both studies. Hepatocytes, isolated by a two-step in situ collagenase perfusion of the liver, were cultured as monolayers and incubated with [14C]diglyme at 1, 10, 30, and 50 μM for up to 48 h. For the in vivo study, rats were given single oral doses of [14C]diglyme at 5.1 mmol/kg body wt, and urine was collected for up to 96 h. Radioactive compounds in the culture medium or in the urine were separated by high performance liquid chromatography and quantified with an in-line radioactivity monitor. Metabolites were identified by comparison of their chromatographic retention times and their mass spectra with those of authentic compounds. The principal metabolite from hepatocytes and in the urine was (2-methoxyethoxy)acetic acid (MEAA). This metabolite accounted for approximately 36% of the radioactivity in the 48-h culture medium and about 67% of the administered dose in the 48-h urine. Other prominent metabolites common to both systems included 2-(2-methoxyethoxy)ethanol, methoxyacetic acid (MAA), 2-methoxyethanol, and diglycolic acid. The diglyme metabolite profiles from urine and from hepatocytes were qualitatively similar, demonstrating that, in the rat, hepatocytes serve as a good model system for predicting the urinary metabolites of diglyme. Moreover, MEAA was shown to be the metabolite best suited for use as a short-term biological marker of exposure to diglyme. Pretreatment of rats with ethanol resulted in a marked increase in the overall in vitro metabolism of diglyme. The major metabolic pathways for diglyme involve O-demethylation and cleavage of the central ether bond, and it is the latter pathway that leads to the formation of MAA, the metabolite associated with the reproductive and developmental toxicity of diglyme. The amounts of MAA formed in hepatocytes from ethanol-pretreated rats ranged from two to four times those formed in hepatocytes from untreated rats.

Alterations in rabbit kidney protein expression following lead exposure as analyzed by two-dimensional gel electrophoresis

It was recently reported that low blood lead levels impaired kidney function in men. To develop a set of molecular markers of renal lead exposure and effect, we investigated changes in renal protein expression while approximating occupational lead exposure at subchronic, low blood levels. Lead was administered to male Dutch Belted rabbits as a lead acetate solution adjusted weekly to achieve and maintain the target blood lead levels of 0, 20, 40, and 80 μg/dL for 15 weeks. Lead exposure did not affect kidney or body weights. The effect of increasing blood lead on protein expression was evaluated in rabbit kidney by large‐scale two‐dimensional electrophoresis (2‐DE). Significant quantitative changes (p < 0.05) occurred in a dose‐related manner in 12 proteins at 20 μg/dL exposure, 25 at 40 μg/dL, and 102 at 80 μg/dL. At a higher level of significance (p < 0.001), 40 μg/dL blood lead resulted in one protein alteration and 80 μg/dL affected 14 proteins. A set of quantitatively altered charge variants was tentatively identified as glutathione‐S‐transferase (GST), based on similar observations in rodents subjected to short‐term, very high lead exposure. The significance of the protein alterations observed as markers of toxicity awaits their conclusive identification. Investigation of the kidney 2‐DE profile in lead‐exposed rabbit may be useful in understanding the mechanism of lead nephrotoxicity in humans.

A Bayesian Network Model for Biomarker-Based Dose Response

A Bayesian network model was developed to integrate diverse types of data to conduct an exposure-dose-response assessment for benzene-induced acute myeloid leukemia (AML). The network approach was used to evaluate and compare individual biomarkers and quantitatively link the biomarkers along the exposure-disease continuum. The network was used to perform the biomarker-based dose-response analysis, and various other approaches to the dose-response analysis were conducted for comparison. The network-derived benchmark concentration was approximately an order of magnitude lower than that from the usual exposure concentration versus response approach, which suggests that the presence of more information in the low-dose region (where changes in biomarkers are detectable but effects on AML mortality are not) helps inform the description of the AML response at lower exposures. This work provides a quantitative approach for linking changes in biomarkers of effect both to exposure information and to changes in disease response. Such linkage can provide a scientifically valid point of departure that incorporates precursor dose-response information without being dependent on the difficult issue of a definition of adversity for precursors.

Assessing Biomarker use in Risk Assessment—A Survey of Practitioners

Advances in molecular epidemiology and mechanistic toxicology have provided increased opportunities for incorporating biomarkers in the human health risk assessment process. For years, the published literature has lauded the concept of incorporating biomarkers into risk assessments as a means to reduce uncertainty in estimating health risk. For all the potential benefits, one would think that markers of effective dose, markers of early biological effects, and markers of human susceptibility are frequently selected as the basis for quantitative human health risk assessments. For this article, we sought to determine the degree to which this evolution in risk assessment has come to pass. The extent to which biomarkers are being used in current human health risk assessment was determined through an informal survey of leading risk assessment practitioners. Case studies highlighting the evolution of risk assessment methods to include biomarkers are also described. The goal of this review was to enhance the implementation of biomarker technology in risk assessment by (1) highlighting successes in biomarker implementation, (2) identifying key barriers to overcome, and (3) describing evolutions in risk assessment methods.

INHIBITION OF RABBIT RENAL ORNITHINE DECARBOXYLASE ACTIVITY BY LEAD

Ornithine decarboxylase (ODC) is the first and the rate-limiting enzyme in polyamine biosynthesis. Polyamines play key roles in cell proliferation, renal membrane transportation, and plasma membrane calcium fluxes. As part of a multidisciplinary research project to evaluate the application of the rabbit as a relevant model for male reproductive toxicology, the effect of blood lead on rabbit renal ODC activity was investigated. Kidneys from rabbits with experimentally established total blood lead concentrations of 0, 20, 40, and 80 mug/dL and maintained at these concentrations for 10 weeks were removed at sacrifice and assayed for ODC activity. ODC activity of rabbit kidney decreased as blood lead concentration increased in a dose-dependent manner. Direct addition of lead to ODC assay mixture did not alter ODC activity. At the blood lead concentrations examined, body weights of each group increased constantly throughout the study period. Overt lead toxicity was not present among the rabbits during the expo...

A Summary of the Workshop "Applying Biomarkers to Occupational Health Practice"

1National Institute for Occupational Safety and Health, Cincinnati, Ohio 2University of ErlangenNuremburg, Erlangen, Germany 3Occupational Safety and Health Administration, Washington, DC 4Johns Hopkins University, Baltimore, Md. 5Rohm and Haas, Co, Spring House, Pa. 6University of Maryland, Baltimore, Md. 7University of Washington, Seattle, Wash. 8Columbia University, New York, N.Y. 9University of Cincinnati, Cincinnati, Ohio 10Sapphire Group, Inc., Washington, DC 11University of Montreal, Montreal, Canada INTRODUCTION

CHARACTERIZATION OF CYTOCHROME P450-DEPENDENT AND GLUTATHIONE TRANSFERASE ACTIVITIES IN SV40-IMMORTALIZED UROEPITHELIAL CELL LINES: POSSIBLE ROLE IN TRANSFORMATION AND TUMOR FORMATION

An in vitro/in vivo transformation system has been developed as a model for bladder tumorigenesis. SV40-immortalized human uroepithelial cells are exposed to putative carcinogens and then implanted into athymic nude mice to testfortumorigenesis.Studieswith4-aminobiphenyl(4-ABP)demonstratedthat one cell line, SV-HUC-PC, was sensitive to chemical-induced transformation and another line, SV-HUC-BC, was refractory.Weare currently testing this system as a model to identify occupational carcinogens and develop biomarkers of exposure and effects of exposure. As part of this study, we examined P450- dependent metabolism, glutathione transferase, and the effects of chemicals on deoxyribonucleic acid(DNA)synthesisandrepair inSV-HUC-PC andSV-HUCBC. Activities for CYP1A1/1A2, CYP3A, and CYP2B1/2B2 were estimated by determining o -dealkylation of ethoxy-, benzoxy-, and pentoxy-resorufin, respectively. Coumarin hydroxylase and p -nitrophenol hydroxylase were used to estimate CYP2A and CYP2E1, respectively. SV-HUC-PC microsomes had fivefold greaterCYP1A1/1A2activityandtwofoldhigherCYP3AactivitythanSV-HUCBC. CYP2B1/2B2 and CYP2A activities and glutathione transferase were not different between the two cell lines. DNA synthesis and repair, by BrdU incorporation, was not different between the two lines when N-methyl-N-nitroN-nitrosoguanidine (MNNG) or other reactive metabolites were tested; however, SV-HUC-PC was more sensitive to n -nitrosodimethylamine, 4-ABP, and 4,4-methylene bis (2-chloroaniline) (MOCA). The data demonstrate that, while these cells have retained form-specific P450 activities, SV-HUC-PC has greater CYP1A1/1A2 and CYP3A activities.