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Entering a new millennium seems a good time to challenge some old ideas, which in our view are implausible, have little supportive evidence, and might best be left behind. In this essay, we summarize a decade of work, raising four issues that involve toxicology, nutrition, public health, and government regulatory policy. (a) Paracelsus or parascience: the dose (trace) makes the poison. Half of all chemicals, whether natural or synthetic, are positive in high-dose rodent cancer tests. These results are unlikely to be relevant at the low doses of human exposure. (b) Even Rachel Carson was made of chemicals: natural vs. synthetic chemicals. Human exposure to naturally occurring rodent carcinogens is ubiquitous, and dwarfs the general public's exposure to synthetic rodent carcinogens. (c) Errors of omission: micronutrient inadequacy is genotoxic. The major causes of cancer (other than smoking) do not involve exogenous carcinogenic chemicals: dietary imbalances, hormonal factors, infection and inflammation, and genetic factors. Insufficiency of many micronutrients, which appears to mimic radiation, is a preventable source of DNA damage. (d) Damage by distraction: regulating low hypothetical risks. Putting huge amounts of money into minuscule hypothetical risks damages public health by diverting resources and distracting the public from major risks.

The mechanism of arsine (AsH3) toxicity is not completely understood. In this investigation, we determined AsH3 and arsenite (AsIII) toxicity in Sprague Dawley rat blood, liver, and kidney. In all systems, there were dose- and time-dependent responses. Red blood cells were very susceptible to AsH3 toxicity. This was demonstrated by an immediate intracellular potassium loss and by hemolysis and lactate dehydrogenase (LDH) leakage that occurred by one h. AsIII concentrations up to 1 mM were not toxic to red blood cells using these indicators. Both AsH3 and AsIII produced toxicity in primary hepatocytes. Both produced significant LDH leakage and decreases in intracellular K+ by 5 h, but AsIII was more toxic than AsH3. At 24 h, both arsenic species showed similar toxicity. In renal cortical epithelial cells, AsH3 produced no effects on LDH and K+ over a 5-h period but produced significant LDH leakage by 24 h. In these cells, AsIII produced significant toxicity as early as in 3 h. These results showed that unchanged AsH3 produced toxicity in tissues, in addition to blood, and that toxicity of arsenicals is arsenic species- and tissue-dependent.

Recent developments in basic immunology have revealed the importance of glutathione (GSH) and cellular redox balance in the generation of an immune response. In the liver, it has been shown that exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) alters cellular GSH and reactive oxygen intermediate (ROI) production. We have tested the hypothesis that TCDD mediates the suppression of the cytotoxic T lymphocyte (CTL) response to alloantigen by increasing oxidative stress. Total cellular GSH, GSSG, and GSH-protein adducts were analyzed by HPLC. Changes in intracellular GSH and ROI were simultaneously measured in isolated hepatocytes and individual subpopulations of spleen cells (CD4+, CD8+, B220+, and Mac-1+) following in vivo exposure to TCDD and antigenic challenge with P815 mastocytoma cells. Monochlorobimane was utilized to measure GSH levels, and two fluorescent probes were used to evaluate ROI levels: dichlorofluoroscein diacetate to monitor peroxides and dihydroethidine to assess superoxide anion. In hepatocytes, in vivo treatment with TCDD resulted in a transient, 2-fold increase in GSH, a 50% decrease in peroxide levels and a small (20-40%) decrease in superoxide anion levels. Although alloantigen challenge resulted in increased GSH and peroxide in spleen cells, in vivo exposure to TCDD had no effect on splenic ROI levels, nor did it consistently alter GSH levels in any subpopulation of spleen cells examined. Moreover, in vivo treatment with the antioxidant N-acetyl cysteine failed to affect the immune suppression caused by TCDD. These results suggest to us that although TCDD perturbs cellular redox balance in the liver, it does not exacerbate or diminish the normal increased GSH and ROI which occur in the spleen in response to antigenic challenge.

Site-specific editing of apolipoprotein B (apoB) mRNA by the cytidine deaminase, APOBEC-1 is proposed to require interactions of auxiliary protein(s) with an eleven nucleotide element, the mooring sequence, located 3' of the C --> U editing site. An analysis of the RNA sequence dependence for protein-RNA interactions and editosome assembly in rat liver and the small intestine demonstrated that the mooring sequence was a minimal requirement for these interactions. Sequences 3' of the mooring sequence either interacted with 66 kDa and 44 kDa proteins or enhanced the interactions of these proteins with the mooring sequence. The data also suggested tissue-specific differences in the relative importance of the 3' cis-acting 'enhancer' elements in the efficiency or stability of editosome assembly. We propose that the previously demonstrated differences in apoB mRNA editing efficiency and its regulation in liver and intestine may in part be due to differences in auxiliary protein interactions with apoB mRNA 3' of the mooring sequence., (Copyright 1999 Academic Press.)

CD154 (CD40 ligand, gp39) interaction with its receptor CD40 has been shown to be critically important for the generation of cell-mediated as well as humoral immunity. It has been proposed that ligation of CD40 on APCs, presumably by activated Th cells, leads to increased APC function as defined by up-regulation of costimulatory molecules and enhancement of IL-12 production. In this report, we directly examined the contribution of the CD154:CD40 pathway in a murine model of allograft rejection. Generation of both the CTL and alloantibody responses following injection with allogeneic P815 tumor cells was severely compromised in CD154 knockout mice and wild-type C57BL/6 mice treated with the anti-CD154 mAb, MR1. Splenic production of IL-2, IFN-gamma, and TNF was significantly suppressed from CD154-deficient mice, indicating a lack of T cell priming. However, splenic cells from CD154 knockout mice induced comparable levels of CD86 expression and IL-12 production when compared with their wild-type littermates. The treatment of CD154-/- mice with the agonistic anti-CD40 mAb, FGK45, generated activated APCs yet failed to restore either the CTL or alloantibody responses to P815. Likewise, immunization with B7-transfected P815 tumor cells failed to generate expansion of the CTL effector population in CD154-/- mice. These results suggest that the generation of allograft immunity is dependent on the interaction of CD154 with CD40 but not primarily for the activation of APCs.

The impact of xenobiotics on intercellular adhesion, a fundamental biological process regulating most, if not all, cellular pathways, has been sparsely investigated. Cell-cell adhesion is regulated in the epithelium primarily by the E-cadherin/catenin complex. To characterize the impact of oxidative stress on the E-cadherin/catenin complex, precision-cut mouse liver slices were challenged with two model compounds for the generation of oxidative stress, diamide (DA; 25-250 microM) or t-butylhydroperoxide (tBHP; 5-50 microM), for 6 h. At the concentrations used, neither compound elicited cytotoxicity, as assessed by intracellular K+ content and leakage of lactate dehydrogenase into the culture media. However, a 25% reduction in non-protein sulfhydryl levels, an indication of oxidative perturbation, was seen in liver slices treated with DA or tBHP. Total protein expression of E-cadherin, beta-, or alpha-catenin was not affected by challenge with DA or tBHP. A decrease of beta-catenin in the SDS-soluble fraction of slices, an indicator of the formation of the adhesion complex, was observed. Additionally, a decrease in beta-catenin interactions with E-cadherin and alpha-catenin, as assessed by immunoprecipitation and Western blot analysis, was seen. Disruption of the E-cadherin/catenin complex by tBHP, but not DA, correlated with enhanced tyrosine phosphorylation of beta-catenin. These results suggest that noncytotoxic oxidative stress disrupts the E-cadherin/catenin cell adhesion complex in precision-cut mouse liver slices.

The interaction of retinoid X receptor alpha with 9-cis-retinoic acid was studied using stopped-flow fluorescence spectroscopy. Transient kinetic analyses of this interaction suggest a two-step binding mechanism involving a rapid, enthalpically driven pre-equilibrium followed by a slower, entropically driven reaction that may arise from a conformational change within the ligand binding domain of the receptor. The assignment of this kinetic mechanism was supported by agreement between the overall equilibrium constant, Kov, derived from kinetic studies with that determined by equilibrium fluorescence titrations. Although these analyses do not preclude ligand-induced alteration in the oligomerization state of the receptor in solution, the simplest model that can be applied to these data involves the stoichiometric interaction of 9-cis-retinoic acid with retinoid X receptor alpha monomers.

The transcription factor, NF-kappaB, is important for T-cell activation, B-cell maturation, and human immunodeficiency virus transcription and plays a role in alternatively mediating and protecting against apoptosis in a variety of cell types. However, a role for NF-kappaB in human CD34(+) bone marrow cells has not been described. We provide evidence here that virtually all human CD34(+) bone marrow cells express NF-kappaB that can be activated by exposure to phorbol 12-myristate 13-acetate and a variety of cytokines, eg, tumor necrosis factor alpha, interleukin-3, and granulocyte-macrophage colony-stimulating factor. In addition, we demonstrate that NF-kappaB may be required for human CD34(+) bone marrow cell clonogenic function and survival. These results offer insight into a new role for NF-kappaB in maintaining survival and function in hematopoietic stem and progenitor cells and suggest that proposed strategies involving inhibition of NF-kappaB activation as an adjunct to cancer chemotherapy should be approached with caution.

Objective: To determine whether an inverse dose-response relationship exists between cigarette smoking and PD among ever-smokers and ex-smokers., Methods: Smoking and alcohol consumption were analyzed in 144 PD patients and 464 control subjects, who were frequency matched for sex, race, and age (+/-5 years), in a population-based case-control study of men and women > or =50 years old in the Henry Ford Health System., Results: With never-smokers as the reference category, there was an inverse association between current light smokers (>0 to 30 pack-years) and PD patients (odds ratio [OR], 0.59; 95% CI, 0.23 to 1.53), and a stronger inverse association of PD with current heavy smokers (>30 pack-years; OR, 0.08; 95% CI, 0.01 to 0.62). When former >30-pack-year smokers were stratified by the interval since quitting, there was an inverse association between those who stopped >20 years ago and PD (OR, 0.86; 95% CI, 0.42 to 1.75), and a greater inverse relationship with those who stopped 1 to 20 years ago (OR, 0.37; 95% CI, 0.19 to 0.72). Alcohol consumption had no independent, significant association with PD, but heavy drinking (>10 drink-years) had a greater effect than light-moderate drinking in reducing but not eliminating the inverse association between smoking and PD., Conclusions: The inverse dose-response relationship between PD and smoking and its cessation is unlikely to be due to bias or confounding, as discussed, providing indirect evidence that smoking is biologically protective.

The effect of different buffer gases on the intensity of negative ions was studied using a gas chromatography/electron monochromator mass spectrometer (GC/EM-MS). The buffer gas was introduced into the ion source not to moderate the electron energies, but specifically to investigate the process of collisional stabilization of negative ions. Three different designs of ion source were tested to study this phenomenon. It was found that collisional stabilization has a profound effect on the intensity of molecular radical anions and begins to play an important role at buffer gas pressures in the order of 10 mTorr. By using a partitioned ion-forming chamber, it was shown that for optimum stabilization to take place the buffer gas should be present in the region of negative ion formation. The gases possessing internal degrees of freedom which are capable of accommodating the excess energy of short-lived excited molecular anion states showed the largest increase in molecular radical anion intensities. At the same time helium, a widely used GC carrier gas, showed sufficient stabilization properties to allow detection of the molecular radical anions of typical electron-capturing molecules with positive electron affinities., (Copyright 1999 John Wiley & Sons, Ltd.)

Denatonium benzoate (DB) is being added to a variety of consumer products as an aversive agent or denaturant. In 1995, Oregon was the first state to mandate the addition of DB to antifreeze and windshield washer fluids regardless of whether the product was marketed in a bottle with a child-proof cap. We have modified published high pressure liquid chromatography (HPLC) methods to measure the concentration of DB and validated it for use in Oregon consumer products. Analysis of standards showed a linear curve in the range of 1.25 to 50 ppm. This makes it useful for most analytical applications as DB usually is added in the 10 to 30 ppm range according to DB's manufacturer. The peak analyzed was collected and verified as DB by mass spectrometry. The method was applied to a variety of commercially-available products with particular emphasis on antifreeze and windshield washer fluids which are required by Oregon law to contain an aversive agent. Products analyzed were purchased in either 1994 or 1996 and showed a wide variation in concentration of DB ranging from below the limit of detection in many of the windshield washer products to above 30 ppm in many of the antifreeze formulations. There was no significant change in levels of DB after storage of antifreeze or windshield washer fluids at room temperature for six months.

The Japanese pufferfish (genus Fugu), which possesses a highly compact genome, is becoming a popular model among those interested in sequencing and mapping the genomes of higher vertebrates. Although genomic libraries have been derived and used to study the molecular biology of Fugu, biological material derived from the living organism is difficult to obtain for laboratories distant from the Asian Pacific. We have established cell cultures from two Fugu species: kusafugu, Fugu niphobles, and torafugu, F. rubripes. Cultures derived from F. niphobles fry and F. rubripes eye have been passaged more than 60 times over the course of one year, representing approximately 180 population doublings. Proliferating cultures were also initiated from F. rubripes brain, liver, fin, spleen, kidney, swimbladder, and muscle. Karyotype analyses indicated that F. rubripes eye-derived cells possessed a chromosome number in the diploid range; F. niphobles fry cells were slightly hyperploid. Flow cytometry confirmed that the relative amounts of DNA present in cultured cells from both Fugu species were similar to that measured in blood cells collected from F. rubripes, and approximately one-seventh of that measured in diploid human cells. Telomerase activity was easily detectable in lysates prepared from F. niphobles fry cells and F. rubripes eye cells, consistent with the notion that these cultures are capable of indefinite proliferation.

Certain polymorphic variants of H-ras-1 and p53 have been investigated for an association between inheritance and cancer risk. The results of a metaanalysis, which reviews studies of H-ras-1 rare alleles and p53 codon 72 allelic variants in breast and lung cancer, are presented. The data constituted evidence for elevated risk of both breast and lung cancer with inheritance of rare H-ras-1 alleles. Calculated population attributable risks are 0.092 and 0.037 for breast and lung cancer, respectively. The frequency of the rare H-ras-1 alleles was observed to be greater in African Americans than in Caucasians, and a specific allele (A3.5) that is common in African Americans was found only at low frequency in Caucasians. For p53 a consensus has yet to be reached. Lung cancer studies conducted in Caucasian and African-American populations have found no evidence of risk associated with the proline variant of codon 72. Two similar studies conducted in Japanese populations suggested an association between p53 genotype distribution and lung cancer risk. However, one implicates the proline allele but the other implicates the arginine allele. The frequency of the proline variant is significantly dependent on race. Frequencies have been reported for control populations of Japanese (0.347 and 0.401), Caucasian (0.295, 0.284, and 0.214), African American (0.628 and 0.527), and Mexican American (0.263).

The ability to culture continuously proliferating cell lines of various organisms in vitro has provided numerous advantages in experimental approaches toward the understanding of basic biology and disease. Although in vitro approaches are common in many disciplines, this methodology has proven difficult to exploit in the study of helminthic parasites. A major cause of parasitic disease, particularly in tropical countries, is the trematode Schistosoma mansoni. We have developed in vitro techniques that allow the long term maintenance of cell cultures from two stages of the life cycle of this organism, associated with its mammalian and the molluscan hosts. We have developed quantitative assays of cell survival and proliferation in our culture systems, and obtained evidence for limited proliferation in vitro. Although the cultures we have achieved thus far are useful for many kinds of experiments in vitro, development of continuously proliferating cell lines remains our goal.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a widespread environmental contaminant and prototypic ligand for the aryl hydrocarbon receptor, is a potent immunotoxicant. To understand the underlying mechanisms of TCDD immunotoxicity, we have characterized the time course of changes in CTL, alloantibody, and cytokine responses to the P815 tumor allograft in C57B1/6 mice treated with 0 or 15 microg TCDD/kg. Suppression of CTL activity by TCDD directly correlated with reduced numbers of splenic CTL effector cells identified by their CD8+CD44 high CD45RB low phenotype, while suppression of the alloantibody response correlated with a lack of expansion of the B220+ splenocyte population. Cytokine production was differentially modulated following TCDD treatment. Although type 1 cytokine production (IFN-gamma, IL-2, and TNF) was initially induced in TCDD-treated mice, production failed to increase normally after day 5. In contrast, the production of IL-1 beta, IL-4, and IL-6 was mostly unaffected by TCDD exposure. This differential effect of TCDD on cytokine production was reflected in the degree of suppression of specific alloantibody isotypes. TCDD abrogated the production of IgG2a (promoted by IFN-gamma), but had much less effect on the level of IgG1 (promoted by IL-4). IgM Ab titers were also highly suppressed. CD8+ cells were the exclusive producers of IFN-gamma and IL-2 when spleen cells from P815-injected mice were cultured in vitro on days 4 to 7 after P815 injection. However, CD4+ cells were shown to play a crucial role in the generation of both CTL and alloantibody responses, since their depletion in vivo abolished both responses. Based on similar temporal effects produced by TCDD and anti-CD4 Ab on alloimmune responses, we postulate that TCDD interferes with the initial activation of CD4+ T cells, which leads to downstream inhibition of the activation and/or differentiation of CD8+ T cells and B cells. In addition, since delayed treatment with either anti-CD4 Ab or TCDD suppressed the alloantibody but not the CTL response, TCDD may also affect later CD4+ T helper-B cell interactions.

HgCl2 is known to be a renal toxin, but its mechanisms of toxicity are not well understood. The cell line LLC-PK1 was used as a model for renal proximal tubule cells, and the effects of different concentrations of HgCl2 were studied. Apoptosis in response to 35 microM HgCl2 was confirmed by observation of morphological features characteristic of apoptotic cells as well as cleavage of chromosomal DNA into fragments of multiples of 200 base pairs. Ten percent of LLC-PK1 cells in a monolayer underwent apoptosis. These cells detached from the culture flask before apoptosis. Measurement of transepithelial resistance (TER) was used as a functional assay of junctional complex integrity in a novel approach to characterize preapoptotic events in this cell line. Monolayers of LLC-PK1 cells that contained apoptotic cells showed a transient decrease in TER followed by a recovery of TER to the initial levels. The decrease in TER was accompanied by a loss of hemicysts within the monolayer. These data indicate a temporary loss of junctional complexes within the monolayer during apoptosis. One hundred micromolar HgCl2 caused all cells to become necrotic within 3 hr. HgCl2 (10 microM) caused some changes in cell morphology, but no cell death.

Although population exposure to lead has declined, chronic lead toxicity remains a major public health problem in the United States affecting millions of children and adults. Important gaps exist in knowledge of the pathophysiology of chronic lead intoxication. These gaps have impeded development of control strategies. To close current gaps in knowledge of chronic lead toxicity, we propose an integrated, multidisciplinary, marker-based research program. This program combines a) direct measurement of individual lead burden by 109Cd X-ray fluorescence analysis of lead in bone, b) determination of ALA-D phenotype, an index of individual susceptibility to lead, and c) assessments of subclinical injury produced by lead in the kidneys, nervous system and, reproductive organs. Data from this research will provide answers to questions of great public health importance: a) Are current environmental and occupational standards adequate to prevent chronic lead intoxication? b) is lead mobilized from the skeleton during pregnancy or lactation to cause fetal toxicity? c) Is lead mobilized from bone during menopause to cause neurotoxicity? d) What is the significance of genetic variation in determining susceptibility to lead? e) What is the contribution of lead to hypertension, renal disease, chronic neurodegenerative disease or declining sperm counts? f) Is chelation therapy effective in reducing body lead burden in persons with chronic overexposure to lead?

Electron monochromators designed for the production of low-energy electrons (0-15 eV) with nearly monoenergetic distributions have been available for many decades. The concept of adapting the electron monochromator as an ion source onto mass spectrometers for the purpose of electron capture negative ion-mass spectrometric (ECNI-MS) analyses is only now being realized. Two different analyzers, a quadrupole and a double focusing sector instrument, have recently been retrofitted with electron monochromators to test their utility as analytical instruments for the detection of environmental compounds and chemical agents. Electron energy scans of compounds in these classes reveal unique negative ion resonances, which can be used as an additional analytical dimension of information for compound identification and confirmation. Electron currents of 430 μA at 0.03 eV electron energy are now available from the electron monochromator, which will provide sufficient electron flux to meet modern standards for trace level analyses. The narrowest electron energy spread achieved has been ±0.07 eV (fwhm). The electron monochromator-mass spectrometer (EM-MS) instrument has been interfaced to a gas chromatograph (GC), and this system (GC/EM-MS) was used to record ion chromatograms of mixtures of polychlorinated compounds. Regioselective ion loss, resulting from dissociative electron capture by the parent molecule, is electron-energy dependent and can be monitored with the EM-MS instrument. Finally, positive ion spectra produced with monoenergetic electrons have also been recorded. © 1997 John Wiley & Sons, Inc., (Copyright © 1997 John Wiley & Sons, Inc.)

Continuous exposure of rats to ozone has been shown to result in lung epithelial damage, inflammation, and subsequent increases in collagen content. The main goal of this study was to identify the earliest time point of altered extracellular matrix protein gene expression by utilizing Northern blot analyses of rat lungs continuously exposed to 1.0 ppm ozone for 14 days. An early increase of steady-state fibronectin mRNA levels was observed at 2 days of exposure, prior to the time point of increased type I collagen mRNA, which was seen at 4 days. This increased level of type I collagen mRNA preceded measurable changes in total lung collagen content, observed at 7 days. In addition, peak levels of the growth-related proto-oncogene c-myc mRNA could be correlated with maximal increases of lung DNA content, although the initial increase in c-myc mRNA preceded measurable changes of total lung DNA. The use of specific cDNA probes for measuring altered gene expression can be useful for defining the early cellular and molecular events in ozone-induced lung injury.

This study sought to determine the selectivity of Pb-induced changes in learning, as distinct from non-specific or performance effects, and to explore the nature of the underlying error patterns contributing to any learning deficits. To accomplish this, rats were chronically exposed to 0, 50, or 250 ppm Pb acetate in drinking water from weaning and trained on a multiple repeated acquisition (RA) and performance (P) schedule beginning at 55 days of age. The RA component required the rat to learn a new 3-member sequence of responses during each experimental session (Center Right Left, RLC, CLR, RCL, and LRC), while the correct sequence of responses for the P component was constant across sessions (LCR). Significant decrements in accuracy on the RA component but not on the P component were found in Pb-exposed groups compared to control, effects that could not be attributed to differential rates of responding. Analyses of error patterns revealed that the effects of Pb exposure on RA accuracy levels derived from two sources. The first consisted of a perseveration of P-like sequence responding (LCR) even during the RA component. Secondly, Pb exposure increased perseverative responding on a single lever, even though the schedule itself never directly reinforced such repetitive responding. The increase in frequency of these two types of perseverative behavior was incompatible with acquisition of non P-like sequences during the RA component. Adding a 5 sec tone to the light stimuli signalling the transition between RA and P components of the multiple schedule failed to attenuate these effects of Pb, suggesting that deficits in stimulus control were not the sole behavioral mechanism of these impairments. Examination of individual data revealed the presence of both 'learners' and 'non-learners' in each group, with the prevalence of the latter being suggestively higher in Pb-exposed groups than in controls. These findings may be relevant to the classroom setting, where periods requiring learning may frequently be interspersed with periods of performance of learned skills.

While several specific genetic alterations have been associated with malignant transformation of human bronchial epithelial cells, they are not all present in every tumor and there is reason to believe that additional genes important for loss of replication control in these cells remain to be identified. In an effort to develop a human bronchial epithelial cell model suitable for identification and functional analysis of genes involved in loss of replication control, and for studying the genetic basis of the multi-stage phenotypic changes associated with tumorigenesis, we treated multiple independent populations of the human papillomavirus (HPV)-immortalized human bronchial epithelial cell line BEP2D with ionizing radiation. Following irradiation, cell lines representing the radiated populations were established from single soft agar-selected colonies. These lines--R2B5SA, R3B5DSA, R2H9S, R2H16S, R2H18S and R3D7S--were compared cytogenetically to the parent cell line and found to have new chromosomal deletions involving putative or confirmed tumor suppressor genes, including chromosome 13 in most R2B5SA cells and all R3B5SA cells, chromosomes 11p and 22 in R216S, and 3p, 11p and 22 in R2H18S. The R2B5SA cells that have lost one copy of chromosome 13 overgrow the culture but are not tumorigenic, suggesting that loss of a single copy of chromosome 13 confers growth advantage but not tumorigenicity. The data confirm that ionizing radiation induces many large chromosomal alterations including chromosomal loss, translocation and deletion and that following radiation it is possible to isolate immortalized nontumorigenic cell lines monosomic for regions known or suspected to contain tumor suppressor genes. The cell lines described here provide powerful models for further investigation of putative tumor suppressor genes including identification, functional analysis and stage of transformation at which they are operative.

In the past, methylmercury compounds were manufactured as fungicides or appeared as unwanted byproducts of the chemical industry, but today the methylation of inorganic mercury in aquatic sediments and soils is the predominant if not the sole source of methylmercury. This form of mercury is bioaccumulated to a high degree in aquatic food chains to attain its highest concentrations in edible tissues in long-lived predatory fish living in both fresh and ocean waters. It is well absorbed from the diet and distributes within a few days to all tissues in the body. It crosses without hindrance the blood-brain and placental barriers to reach its principal target tissue, the brain. It is eliminated chiefly in the feces after conversion to inorganic mercury. The biological half-time of methylmercury in human tissues is about 50 days, but there is wide individual variation. Adult poisoning is characterized by focal damage to discrete anatomical areas of the brain such as the visual cortex and granule layer of the cerebellum. A latent period of weeks or months may ensue before the appearance of signs and symptoms of poisoning. The latter manifest themselves as paresthesia, ataxia, constriction of the visual fields, and hearing loss. The prenatal period is the most sensitive stage of the life cycle to methylmercury. Prenatally poisoned infants exhibit a range of effects from severe cerebral palsy to subtle developmental delays. Methylmercury is believed to inhibit those processes in the brain specially involved in development and growth such as neuronal cell division and migration.

Exposure to lead (Pb) has been reported to inhibit MK-801 binding and to alter other NMDA receptor complex-associated functions. These reported changes are provocative since both NMDA receptor antagonism and Pb exposure are know to impair learning processes. Whether the Pb-induced changes in NMDA function relate to the learning impairments associated with Pb exposure, however, has not been explored. The contention of this study was that if changes in NMDA function produced by Pb serve as the basis of Pb-associated learning impairments, then such changes should be of sufficient biological magnitude and clinical relevance to induce alterations in sensitivity at the level of the whole animal, i.e., changes in behavioral sensitivity to glutamatergic compounds. Thus, in this study, dose-effect curves of control and Pb-treated rats working on a multiple schedule of repeated learning (repeated acquisition, RA) and performance (P) were compared following acute administration of MK-801, the non-competitive NMDA antagonist. Based on the nature of the reported effects of Pb on NMDA systems, it was expected that the curves of Pb-exposed rats would be right-shifted relative to controls, if differential behavioral sensitivity was evident. Rats were chronically exposed to 0, 50 or 250 ppm Pb acetate in drinking water from weaning and trained on the multiple RA and P schedule beginning at 55 days old. The RA component required the rat to learn a new 3-member sequence of responses during each experimental session (center right left, RLC, CLR, RCL, or LRC), while the correct sequence of responses for the P component was constant across sessions (LCR), requiring performance of an already learned response. Acute administration of MK-801 (0.05-0.3 mg/k, i.p.) resulted in decrements in accuracy in both the RA and P components of the schedule, indicative of non-specific effects on behavior rather than selective effects on learning. The declines in accuracy during the RA component of the schedule were primarily the result of increased perseverative responding, i.e., repetitive responding on a single lever. Both the decline in RA accuracy and the increases in perseverative responding produced by MK-801 were attenuated by Pb exposure. Moreover, dose-effect curves relating MK-801 dose to changes in rates of responding were significantly shifted to the right in Pb-exposed rats relative to controls. Taken together, these data demonstrate a subsensitivity of Pb-exposed rats to both the accuracy-impairing and response rate-altering properties of MK-801.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat pheochromocytoma PC12 cell sublines with increasing resistance to methylmercury (MeHg) were isolated by exposure to MeHg in two sequential steps. The strongest resistance (PC12/TM) among the clones obtained by the first cloning was characterized. PC12/TM cells exhibited about an 8- to 10-fold increase in resistance compared with parent PC12 cells on the bases of the concentration required for 50% inhibition of growth and colony-forming activity. PC12/TM cells accumulated smaller amounts of MeHg (one-half to one-fifth) than parent PC12 cells. This reduced MeHg accumulation in PC12/TM cells resulted from the slow uptake and rapid efflux. A close correlation between reduced MeHg accumulation and MeHg resistance was found among seven sublines of PC12 cells with different sensitivity to MeHg. The reduced retention of MeHg in PC12 sublines was also well correlated with the sensitivity to MeHg. Phenol-3,6-dibromophthalein disulfonate inhibited MeHg efflux from PC12/TM cells and increased its accumulation. These results suggest that efflux of MeHg from PC12/TM cells is associated with glutathione.

To examine the impact of lead (Pb) exposure during the ontogeny of dopaminergic (DA) systems on resultant DA function, rats were exposed postnatally (0-21 days of age) via the lactating dam to 0, 100 or 350 ppm Pb acetate in drinking water. At 2 months of age, the postnatally Pb-exposed rats were trained to discriminate the stimulus properties of either the D1 receptor agonist SKF38393 (6.0 mg/kg) or the D2-D3 receptor family subtype agonist quinpirole (0.05 mg/kg) from saline using a standard two-lever operant food-reinforced drug discrimination paradigm. In each training group, dose-effect curves describing drug lever responding to lower doses of the training drug and to preadministration of selective DA antagonists were obtained to examine Pb-induced changes in DA sensitivity, and doses of non-DA compounds were substituted to determine the specificity of any changes in DA sensitivity. In the D1/saline training condition, Pb exposure was not associated with any specific or consistent changes in DA sensitivity. In contrast, exposure to Pb was associated with D2-D3 receptor subtype supersensitivity as was indicated by significantly elevated levels of drug lever responding in the presence of quinpirole and haloperidol and to at least one dose of apomorphine. No differences in the dose-effect curves for either (+)-amphetamine or NMDA were observed in the D2-D3-trained control and Pb-exposed groups, but an increase in drug lever responding in the presence of pentobarbital was noted in the Pb-exposed group relative to control. Taken together, these findings are consistent with a Pb-induced functional D2-D3 supersensitivity possibly mediated via autoreceptors. Moreover, this functional D2-D3 supersensitivity necessarily represents a permanent effect of postnatal Pb exposure since both blood and brain Pb levels were negligible at the time drug discrimination training began.

The ability of synthetic metalloporphyrins to suppress heme oxygenase activity and bilirubin formation has recently become of considerable clinical and experimental interest for suppression of jaundice in humans, including neonatal hyperbilirubinemia. The present investigation compares the biochemical effects of Sn- and Zn-protoporphyrins on the predominant heme oxygenase isozyme present in the brain (HO-2) at activity, protein, and transcript levels and describes the ability of Sn-protoporphyrin to adversely affect this isozyme. Specifically, 6 h after a modest dose (50 mumol/kg, i.v.) of Sn-protoporphyrin, heme oxygenase activity in rat brain was nearly undetectable. In addition, as revealed by Western blot analysis, HO-2 protein level was decreased by 20% and the electrophoretic behavior of the protein in the microsomal membranes was altered. Moreover, the activity of NADPH-cytochrome P-450 reductase, which is required for the oxidation of heme molecule, was markedly decreased (60% of control). Western immunoblot analysis revealed also a pronounced decrease in the reductase protein level. The inducible form of heme oxygenase, HO-1, was not detectable by immunoblotting in brain microsomes of either control or Sn-protoporphyrin-treated animals. Northern blot analyses did not reveal decreases in the levels of the single HO-1 mRNA (1.8 kb) or the two HO-2 transcripts (1.3 and 1.9 kb), suggesting that Sn-protoporphyrin mediates its effects on heme oxygenase isozymes at the protein level. Zn-protoporphyrin, on the other hand, had no deleterious effect on brain parameters presently investigated.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigations into the cause of health problems on a horse-breeding farm led to the discovery of high concentrations (630 to 9,810 mg/kg of bedding) of pentachlorophenol in wood shavings used as bedding for horses over a period of 2 to 4 years. Toxicologic signs in the horses were characteristic of toxic effects associated with exposure of polychlorinated dibenzo-p-dioxins and dibenzofurans. Tissue residue analysis confirmed presence of toxic polychlorinated dibenzo-p-dioxin and dibenzofuran isomers known to be in pentachlorophenol, substantiating the bioavailability of polychlorinated dibenzo-p-dioxins and dibenzofurans in the wood shavings. The findings provide evidence that residue concentrations in the range of 2 ng/g of toxic polychlorinated dibenzo-p-dioxin isomers in liver or fat correlate with toxicologic effects in horses.

We conducted a series of experiments with ultrafine particles (approximately 20 nm) and larger particles (less than 200 nm) of "nuisance" dusts to evaluate the involvement of alveolar macrophages (AM) in particle-induced lung injury and particle translocation in rats. After intratracheal instillation of both ultrafine particles and larger particles of TiO2, we found a highly increased interstitial access of the ultrafine particles combined with a large acute inflammatory reaction as determined by lung lavage parameters. An additional experiment revealed that intratracheal instillation of phagocytized ultrafine TiO2 particles (inside AM) prevented both the pulmonary inflammatory reaction and the interstitial access of the ultrafine particles. Another experiment showed that the influx of polymorphonuclear cells (PMN) into the alveolar space unexpectedly decreased with higher doses of ultrafine particles, whereas alveolar epithelial permeability (protein leakage) increased. The divergence between PMN influx into the alveolar space and changes in alveolar epithelial permeability implies that they are separate events. Pulmonary inflammatory parameters determined by lung lavage analysis correlated best with the surface area of the retained particles rather than with their mass, volume, or numbers. Because higher doses resulted in an increased interstitialized fraction of particles, we suggest that inflammatory events induced by particles in the interstitial space can modify the inflammation in the alveolar space detectable by lung lavage. Our results demonstrate the dual role of AM for modifying particle-induced lung injury, i.e., both preventing such injury and contributing to it. We conclude that the increased pulmonary toxicity of ultrafine particles is related to their larger surface area and to their increased interstitial access.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Lead (Pb) is a ubiquitous environmental toxicant which has been reported to have growth-retarding effects. That premise was examined in the current study of the effects of developmental exposure to Pb on the maturation of the fruitfly Drosophila melanogaster. 2. Flies were raised from egg to adulthood in media to which 0, 100, 250, or 500 ppm Pb acetate added. 3. There was a dose-dependent delay of maturation but no apparent effect on survival to adulthood. There were no significant differences in this effect between male and female flies. 4. Weights of fly offspring and their fecundity were not related to increasing exposure levels, suggesting that the delays were not due to gross nutritional deficits. 5. Analyses of Pb content indicated exposure-dependent body burdens of Pb in flies. Timed analyses of the Pb content of media itself indicated a heterogeneous distribution of Pb in the media, suggesting some precipitation of Pb at the highest exposure, occurring primarily during the first hour. 6. The mechanistic bases of the Pb-induced retardation of D. melanogaster development remain unknown, but it is concluded that because of the extensive body of knowledge on D. melanogaster genetics, molecular biology, and developmental biology, this procedure could serve as a model system for further study of the developmental consequences of exposure to Pb or other toxicants. 7. Environmental Pb exposure resulting in retarded development could have deleterious repercussions for insect populations exposed chronically to high levels of Pb.

Manganese produces signs and symptoms that suggest involvement of the basal ganglia, especially the globus pallidus and substantia nigra. Overt neurological signs have been reported in primates exposed to high levels of manganese (over 100 mg/kg) but little is known about the effects of lower doses. To examine these issues, three cebus monkeys were trained to operate a response device with their arms and legs by executing a rowing-like movement against a 3.9- to 4.1-kg spring through an arc length of 10 cm under a multiple fixed-ratio fixed-interval schedule of reinforcement. Over the course of 450 days, these monkeys were administered acute doses of 5 or 10 mg/kg iv of manganese chloride using a multiple baseline experimental design. Doses as low as 5 mg/kg provoked a large increase in the number of incomplete responses. The onset of manganese's effect appeared within days of exposure and developed over the course of several weeks. Its magnitude declined over the course of months, but after a cumulative dose of 10 to 40 mg/kg it did not return to baseline. Action tremor appeared at cumulative doses greater than 40 mg/kg and dystonia was never observed at the cumulative doses examined. Behavioral microanalysis revealed that manganese's effects initially appeared as increased variability of interresponse times and response duration. Later, the response pattern during the fixed ratio component shifted to one of progressively increasing durations through the course of the ratio. Magnetic resonance imaging revealed that the behavioral effects of manganese corresponded to an apparent increase in the manganese content of the globus pallidus and substantia nigra.

Exposure to thermal degradation products arising from fire or smoke could be a major concern for manned space missions. Severe acute lung damage has been reported in people after accidental exposure to fumes from plastic materials, and animal studies revealed the extremely high toxicity of freshly generated fumes whereas a decrease in toxicity of aged fumes has been found. This and the fact that toxicity of the freshly generated fumes can be prevented with filters raises the question whether the toxicity may be due to the particulate rather than the gas phase components of the thermodegradation products. Indeed, results from recent studies implicate ultrafine particles (particle diameter in the nm range) as potential severe pulmonary toxicants. We have conducted a number of in vivo (inhalation and instillation studies in rats) and in vitro studies to test the hypothesis that ultrafine particles possess an increased potential to injure the lung compared to larger-sized particles. We used as surrogate particles ultrafine TiO2 particles (12 and 20 nm diameter). Results in exposed rats showed that the ultrafine TiO2 particles not only induce a greater acute inflammatory reaction in the lung than larger-sized TiO2 particles, but can also lead to persistent chronic effects, as indicated by an adverse effect on alveolar macrophage mediated clearance function of particles. Release of mediators from alveolar macrophages during phagocytosis of the ultrafine particles and an increased access of the ultrafine particles to the pulmonary interstitium are likely factors contributing to their pulmonary toxicity. In vitro studies with lung cells (alveolar macrophages) showed, in addition, that ultrafine TiO2 particles have a greater potential to induce cytokines than larger-sized particles. We conclude from our present studies that ultrafine particles have a significant potential to injure the lung and that their occurrence in thermal degradation events can play a major role in the highly acute toxicity of fumes. Future studies will include adsorption of typical gas phase components (HCl, HF) on surrogate particles to differentiate between gas and particle phase effects and to perform mechanistic studies aimed at introducing therapeutic/preventive measures. These studies will be complemented by a comparison with actual thermal degradation products.

Several haloalkenes are selective nephrotoxins. The bioactivation of nephrotoxic haloalkenes involves hepatic glutathione S-conjugate formation, peptidase-catalyzed metabolism of the glutathione S-conjugates to the corresponding cysteine S-conjugates, uptake of cysteine S-conjugates by the kidneys, and renal cysteine conjugate beta-lyase-catalyzed beta-elimination of a thiol. The haloalkyl and haloalkenyl thiols thus released are unstable and yield reactive intermediates whose interactions with cellular constituents are though to contribute to the observed toxicity of S-conjugates. Tetrafluoroethene and chlorotrifluoroethene are metabolized to the cysteine S-conjugates S-(1,1,2,2-tetrafluoroethyl)-L-cysteine (TFEC) and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine (CTFC), respectively. Administration of TFEC (1.0 mmol/kg) or CTFC (1.0 mmol/kg) to rats resulted in acylation of renal proteins, as demonstrated with 19F nuclear magnetic resonance spectroscopy. Single, broad resonances near 41 or 56 ppm were found in spectra of renal proteins from TFEC- or CTFC-treated rats, respectively, and these resonances were not lost on dialysis. Renal protein incubated with 2-chloro-1,1,2-trifluoroethyl-2-nitrophenyl disulfide, a proreactive intermediate that yields 2-chloro-1,1,2-trifluoroethanethiol, showed the same 19F NMR spectrum as was found with CTFC-treated rats. In vitro incubation of various N alpha-blocked amino acids with this proreactive intermediate indicated that only lysine is stably adducted, whereas histidine is transiently acylated. In each case, proteolysis of modified protein converted a single broad NMR resonance to a doublet with little change in chemical shift and with clearly resolved, characteristic H-F couplings. The single, stable amino acid adduct formed with renal proteins of rats given CTFC or TFEC was N epsilon-(chlorofluorothioacetyl)lysine and N epsilon-(difluorothioacetyl)lysine, respectively.

The factors affecting the deposition of inhaled cadmium particles in different regions of the respiratory tract are discussed. The retention kinetics of inhaled cadmium compounds is determined by the various clearance processes. It is necessary to differentiate clearly between in vitro (water) solubility and in vivo (lung) solubility. Thus both cadmium oxide and cadmium sulfide particles are rather insoluble in water but the former are readily solubilized in the lung while the latter are cleared mainly by mechanical transport via alveolar macrophages. The pulmonary retention half-times of inhaled cadmium compounds are longer by approximately a factor of ten in primates than in rats. Acute pulmonary inflammatory effects of inhaled cadmium compounds are related to their in vivo solubility, and an approximately 10-fold higher dose of cadmium sulfide as compared with the chloride or oxide must be administered to the lung to elicit the same inflammatory response. Differences in biological effects may also exist with respect to the chronic effects of inhaled cadmium compounds, e.g., the pulmonary carcinogenic potency of inhaled cadmium sulfide may be less than that of other cadmium compounds.

An understanding of the differential role of cholinergic and glutaminergic systems may be limited by the failure to move the analysis of learning impairments beyond an assessment of changes in overall accuracy. This paper reports the results of two studies in which the effects in rats of scopolamine (0.5-3.0 mg/kg IP), a cholinergic antagonist, and MK-801 (0.05-0.3 mg/kg IP), an NMDA-receptor antagonist, were compared in two different repeated learning procedures and the nature of the underlying error patterns produced by each was evaluated. The first study examined drug effects upon a repeated sequence acquisition procedure and found that while both drugs decreased overall accuracy in a dose-dependent manner, the predominant error pattern varied significantly with drug; scopolamine primarily produced skipping errors within the sequence, whereas MK-801 more prominently increased perseveration on the first and second members of the sequence. In the second study, which used a repeated transition procedure, both drugs again significantly decreased overall accuracy in a dose-dependent manner, but no consistent differences in error patterning produced by the drugs were observed. Thus, while both cholinergic and NMDA systems play a role in learning, the behavioral processes underlying the changes in overall accuracy may differ, as indicated by the differential patterns of errors produced by scopolamine and MK-801 in the repeated acquisition baseline. Furthermore, the observed differences in the underlying behavioral processes of scopolamine and MK-801 in the repeated acquisition but not on the repeated transition procedure suggest that each of the two drugs may affect more than one of the variables controlling behavior, with the relative impact of drug-related changes in controlling variables depending upon the operative contingencies of the learning task.

Since both Pb exposure and aging have been associated with alterations in memory functions, this study compared the effects of Pb exposure initiated at early, middle and later stages of the life cycle on delayed spatial alternation performance. Young (21 day old), adult (8 mon old) and old (16 mon old) rats were exposed for a total duration of 8.5 mon to 0, 2 or 10 mg Pb acetate/kg/day (young rats) or 0, 1.9 or 9.3 mg Pb acetate/kg/day (adult and old rats) in drinking water. These doses were designed to produce equivalent brain Pb concentrations across the three age groups. Behavioral testing began after 4 mon of exposure utilizing a standard delayed alternation paradigm with delay values of 0, 3, 6, 9 and 12 sec presented randomly during each experimental session for a total of 40 sessions. Aging, as expected, was associated with impairments of accuracy, but the disruption of accuracy at the 0 sec delay suggested that these were performance rather than memory deficits. Pb exposure actually improved delayed spatial alternation performance in both young and old animals: accuracy values were increased, while error frequencies declined in the two age groups. The increases in accuracy occurred primarily at the longest delay value in young rats, but were more prominent at short delay values in old rats. This difference may have been the result of a ceiling effect, since accuracy for young rats at short delay values was already quite high. Delayed spatial alternation performance of adult rats was generally unaffected by Pb exposure. A possible explanation of the improved performance of young and old Pb-exposed rats is one based on response perseveration, i.e., a facilitation of rote alternation behavior engendered by the cued alternation training program. The results of this study suggest that both early and late stages of the life cycle must be considered periods of enhanced vulnerability to Pb-induced behavioral changes.

While early development represents a period of particular sensitivity to the neurotoxic effects of lead (Pb), much less is known about CNS effects at later stages of the life cycle. It is conceivable that old age represents an additional period of vulnerability given the neurodegenerative processes that are the inevitable accompaniment of advanced age. The experiments described here examined that possibility. The effects of prolonged low-level Pb exposures were assessed in young (21 day old), adult (8 mon old) and old (16 mon old) F-344 rats performing on Fixed-Interval (F1) and Variable-Interval (VI) schedules of reinforcement, two behavioral baselines previously shown to be sensitive to Pb exposure in young rats. The profile of Pb effects within each age group was remarkably uniform across both reinforcement schedules, while the direction of effect differed by age group. Pb exposure was associated with shifts toward increased VI and FI response rates in both young and old rats. In contrast, adult rats exposed to Pb exhibited decreased rates of responding on both FI and VI schedules. Both patterns of response rate changes can be indicative of less efficient performance. Of the two reinforcement schedules, the FI appeared to be the more sensitive, as marginally significant behavioral effects were observed at both the 2 and 10 mg Pb/kg/day doses on the FI, while only occurring in response to the 10 mg/kg dose of Pb on the VI. The differential pattern of Pb effects across age groups does not appear to reflect a rate-dependency phenomenon, but its exact basis awaits further investigation. The detection of primarily marginally significant response rate changes here as compared to previous studies using similar Pb exposure levels suggests that the F-344 strain of rats is less sensitive to Pb than is the Long-Evans rat strain.

The aryl hydrocarbon (Ah) receptor undergoes a ligand-dependent transformation to a heteromeric structure which has the ability to bind DNA sequence-specifically with high affinity. By this mechanism, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related xenobiotics modify gene expression. We observed that transformation was inhibited in vitro by the presence of ribonuclease A (RNAase) during incubation of rat hepatic cytosol with TCDD. This effect was detected as a decreased ability of the TCDD-receptor complex to bind to calf thymus DNA covalently linked to Sepharose, and to a dioxin-responsive enhancer which is upstream of the cytochrome P450IA1 structural gene. RNAase had no effect on previously transformed TCDD-receptor complexes. These observations indicated that RNAase acted during ligand binding and/or transformation of the Ah receptor. Saturation binding analyses demonstrated that RNAase decreased the receptor affinity for TCDD without changing the total number of binding sites. RNAase also inhibited transformation of the TCDD-bound, partially purified, untransformed, receptor. Thus RNAase does not interfere with ligand binding, but inhibits the subsequent transformation of the receptor monomer to the heteromeric, transcriptionally active, form.

The status of glutathione (GSH) was studied in isolated rat liver mitochondria under conditions which induce a permeability transition. This transition, which is inhibited by cyclosporin A (CyA), requires the presence of Ca2+ and an inducing agent such as near physiological levels (3 mM) of inorganic phosphate (Pi). The transition is characterized by an increased inner membrane permeability to some low molecular weight solutes and by large amplitude swelling under some experimental conditions. Addition of 70 microM Ca2+ and 3 mM Pi to mitochondria resulted in mitochondrial swelling and extensive release of GSH that was recovered in the extramitochondrial medium as GSH. Both swelling and the efflux of mitochondrial GSH were prevented by CyA. Incubation of mitochondria in the presence of Ca2+, Pi, and GSH followed by addition of CyA provided a mechanism to load mitochondria with exogenous GSH that was greater than the rate of uptake by untreated mitochondria. Thus, GSH efflux from mitochondria may occur under toxicological and pathological conditions in which mitochondria are exposed to elevated Ca2+ in the presence of near physiological concentrations of Pi through a nonspecific pore. Cyclical opening and closing of the pore could also provide a mechanism for uptake of GSH by mitochondria.

The term neurodegenerative denotes a process rather than a state. In contrast, most research on such disorders, whether clinical or experimental, represents only a slice of time. Their progressive nature is mostly confined to speculation instead of being codified in research protocols. Research on cancer, also a degenerative disease, is much more often framed in terms of process. Part of the difference is accounted for by the impact of quantitative modeling, which enjoys a long history in both basic and clinical cancer research and which originated in attempts to describe and understand tumor development. Analogous questions are posed by neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease. Among the issues that modeling could help to clarify are the properties and sources of the age-specific incidence rate, which resembles that for cancer, and the pharmacokinetics governing the toxic products of neurotransmitter metabolism. Neurodegenerative disorders maintain a research advantage because functional measures, mostly inaccessible to cancer investigators, serve as the ultimate index of progression. Their exploitation, however, in longitudinal studies remains inadequate.

As a model of non-immunological lung inflammation, guinea pigs were exposed to 2.0 ppm ozone for 4 hours. Polymorphonuclear leukocytes (PMNs) rapidly accumulated in lung interstitium but declined from 147 +/- 34 million cells to control values (33 +/- 6 million cells) within the first 24 hours. Bronchoalveolar lavage (BAL) recovered PMNs were maximal by 3-6 hours (4 +/- 1 million cells) and remained elevated for 3 days. Macrophage numbers were doubled in lavageable spaces but those in interstitium increased by only a third at 2 days post-exposure. By 7 days post-exposure BAL macrophages had declined to control values of 12 +/- 2 million cells while those in interstitium remained elevated through 14 days. These data demonstrate that BAL does not necessarily reflect cellular changes in lung interstitium.

Although the nucleotide bases that constitute the consensus DNA sequence of the estrogen response element (ERE) have been identified, the involvement of electrostatic contacts between the sugar-phosphate backbone of the ERE and the estrogen receptor (ER) is not known. Moreover, the contribution of these contacts to sequence-specific DNA binding has not been determined. Therefore, the interactions of highly purified ER with the phosphate residues of the ERE derived from the chick vitellogenin (cVit)-II gene were examined by phosphate ethylation interference. Specific ER-DNA complexes were evident in electrophoretic gel mobility shift assays using DNA fragments containing either the perfect ERE (-625 relative to gene start site; 5'-GGTCAGCGTGACC) or the imperfect ERE (-353; 5'-GGTCAACATAACC). The phosphate ethylation interference footprint identified a 2-fold, symmetrical exclusion of phosphate residues essential for specific binding to the perfect ERE with a 5' stagger, indicating that each monomer of the ER dimer is bound in the major groove of the DNA. The interference footprint of the imperfect ERE did not detect interactions between the receptor and the phosphate residues in the 3' half of the response element on the noncoding strand. In contrast, the corresponding footprint of the perfect ERE displayed strong interactions between the ER and the phosphate backbone of the DNA. Consequently, the absence of these electrostatic contacts very likely accounts for the reduced binding affinity of the ER for the imperfect ERE. These results indicate that specific contacts between the ER and the sugar-phosphate backbone of its cognate response elements are an important aspect of DNA sequence recognition and high affinity binding.

When treated with the cytosolic Ca++ indicator Quin 2-acetoxymethyl ester (Quin 2-AM), isolated hepatocytes exhibited signs of toxicity, such as extensive lipid peroxidation and vitamin E loss and release of lactate dehydrogenase. Lipid peroxidation induced by this agent was blocked completely by cotreatment of the cells with ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid, EDTA, ruthenium red, carbonyl cyanide m-chlorophenylhydrazone, desferal and trifluoperazine, and was partially inhibited by quinacrine and indomethacin. With the exception of carbonyl cyanide m-chlorophenylhydrazone and quinacrine, these agents also inhibited lactate dehydrogenase leakage. Although the results with ruthenium red suggested that Quin 2-AM may cause toxicity by altering handling of Ca++ by mitochondria, mitochondrial membrane potential was not altered in cells treated with Quin 2-AM until after toxicity occurred. Evidence of a direct, potentiative effect of Quin 2 on iron-induced lipid peroxidation was gained from experiments with liposomes. Treatment of cells with Quin 2-AM did not enhance nitro blue tetrazolium reduction, suggesting that Quin 2 did not stimulate O2- production by the cells. Direct chelation of Ca++ did not appear to be involved in the mechanism of Quin 2 toxicity, for an analog of Quin 2 that is virtually nonhydrolyzable, which greatly limits the binding of Ca++, also caused lipid peroxidation and cell death. These results suggest that Quin 2 causes toxicity by chelating iron or by activating some cellular process(es) that is dependent on the presence of iron or Ca++.

The Ah receptor binds 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related aryl hydrocarbons and mediates their biochemical and toxic effects by modifying gene expression. In order to interact with DNA, the TCDD.receptor complex must undergo a poorly understood transformation to a form which is distinguishable by its increased affinity for DNA-Sepharose and for its specific enhancer element upstream from the cytochrome P450IA1 gene. We have found that this transformation process is inhibited in vitro by treatment of rat hepatic cytosol with activated charcoal prior to addition of [3H]TCDD. The transformed form of the receptor can be generated in the charcoal-inhibited cytosol by incubation with hepatic cytosol from either DBA/2J mice (in which [3H]TCDD-specific binding is undetectable under these conditions) or rat (in which Ah receptor was prebound with unlabeled ligand). Transformation is observed whether this addition occurs before or after [3H]TCDD is bound to the charcoal-treated receptor. Thus, transformation is (i) mediated by some additional cytosolic component(s) and (ii) separable from ligand binding. The untransformed [3H]TCDD.receptor complex, isolated by DNA-Sepharose chromatography, can also be transformed if DBA mouse hepatic cytosol is added. This partially purified untransformed receptor preparation and gel retardation analysis were used to further characterize the transforming activity in DBA cytosol. We observed that the "Ah receptor transforming factor" is heat-labile, trypsin-sensitive, removed or inactivated by charcoal, of greater than approximately 50 kDa, and elutes from Superose at a Rs of approximately 6 nm. In conjunction with our previous studies documenting the increased molecular weight of the transformed compared to the untransformed Ah receptor, and identifying the heteromeric structure of the transformed receptor, we postulate that the ligand-binding subunit (the untransformed receptor) must associate tightly with another cytosolic protein, which is also present in the receptor-defective DBA mouse, in order to transform to the transcriptionally active receptor.

1,2-Dichloro-1,1-difluoroethane (HCFC-132b) is a potential substitute for some ozone-depleting chlorofluorocarbons and a model for other 1,1,1,2-tetrahaloethanes under consideration as chlorofluorocarbon substitutes. Male Fischer 344 rats were given 10 mmol/kg HCFC-132b dissolved in corn oil by intraperitoneal injection. An NMR assay for covalent binding of HCFC-132b metabolites to liver proteins was negative, whereas binding was observed in halothane-treated rats. Total urinary metabolites excreted by rats given HCFC-132b during the first 24 h amounted to 1.8 +/- 0.1% of the injected dose, as determined by 19F NMR. During the first 6 h, metabolites of HCFC-132b corresponding to 2-chloro-2,2-difluoroethyl glucuronide, unknown metabolite A, chlorodifluoroacetic acid, and chlorodifluoroacetaldehyde hydrate [both free and conjugated (unknown metabolite B)] were excreted in urine in the approximate ratio 100:9:3:7, respectively. Metabolite A is apparently an O-conjugate of 2-chloro-2,2-difluoroethanol; unconjugated 2-chloro-2,2-difluoroethanol was not detected in urine. The 19F NMR spectrum of metabolite B indicates the formation of a hemiacetal of chlorodifluoroacetaldehyde. Repeated exposure of rats to HCFC-132b significantly increased both the rate of chlorodifluoroacetic acid excretion and the relative fraction of the HCFC-132b dose excreted as chlorodifluoroacetic acid in urine. Incubation of HCFC-132b with rat hepatic microsomes yielded chlorodifluoroacetaldehyde hydrate as the only fluorinated product. The in vitro metabolism of HCFC-132b was increased in microsomes from pyridine-treated rats as compared with control rats, and HCFC-132b metabolism was inhibited by p-nitrophenol, indicating that the cytochrome P-450 isoform IIE1 is largely responsible for the initial hydroxylation of HCFC-132b.

Studies were carried out to investigate the metabolism of senecionine by human liver microsomes and the role of human cytochrome P450IIIA4 in this process. Human liver microsomes metabolized senecionine to two major products, (+/-)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) and senecionine N-oxide. The rates of product formation (DHP and senecionine N-oxide) varied widely with the microsomal samples tested. There was a 30-fold difference in DHP formation and a 25-fold difference in N-oxidation between the poorest metabolizer and the highest metabolizer of senecionine. The conversion of senecionine to DHP and senecionine N-oxide in human liver microsomes was markedly inhibited by the mechanism-based inactivators of P450IIIA4, gestodene and triacetyloleandomycin. Anti-P450IIIA4 IgG, at a concentration of 1 mg/nmol of P450, was found to inhibit completely the formation of DHP and senecionine N-oxide in human liver microsomes (HL101) having low activity toward senecionine. At 5 mg IgG/nmol P450, anti-P450IIIA4 inhibited 90 and 84% respectively of the formation of DHP and senecionine N-oxide in liver microsomes (HL110) with the highest activity toward senecionine. The formation of DHP or senecionine N-oxide was highly correlated with the amount of P450IIIA4 measured in the microsomes using polyclonal anti-P450IIIA4 IgG. The rate of DHP production also had a strong correlation with the rate of senecionine N-oxide formation (r = 0.999) and with the rate of nifedipine oxidation (r = 0.998). Our present studies provide evidence that P450IIIA4 is the major enzyme catalyzing the bioactivation (DHP formation) and detoxication (senecionine N-oxide formation) of senecionine in human liver.

Information is presented on the impact of land application of municipal sewage sludge on the selenium content and speciation in soil, groundwater and edible vegetation. Sources and typical concentrations of selenium in sludge are documented. A discussion of selenium uptake by agricultural crops from sludge-amended soil includes results from greenhouse and field studies. A comparison is made with crop selenium uptake from fly ash application. The effect of sludge treatment on animal and human dietary selenium intake is quantitatively evaluated and selenium guidelines for sludge application are summarized. The conclusion is made that future widespread use of sludge on agricultural land will result in increased selenium uptake by food crops and human dietary intake. While this may not present an increased human health risk, long-term risks are identified and recommendations are made to minimize them.