High-affinity renal lead-binding proteins in environmentally-exposed humans.

Chronic low level lead (Pb) exposure is associated with decrements in renal function in humans, but the molecular mechanisms underlying toxicity are not understood. We investigated cytosolic Pb-binding proteins (PbBP) in kidney of environmentally-exposed humans to identify molecular targets of Pb and elucidate mechanisms of toxicity. This study is unique in that it localized PbBPs based on physiologic Pb that was bound in vivo. Two Pb-binding polypeptides were identified, thymosin beta 4 (T beta 4, 5 kDa) and acyl-CoA binding protein (ACBP, 9 kDa, also known as diazepam binding inhibitor, DBI). These polypeptides, which have not been previously recognized for their metal-binding capabilities, were shown to bind Pb with high affinity (Kd approximately 14 nM) and to account for an estimated > 35% of the total Pb in kidney cortex tissue. Both T beta 4 and ACBP (DBI) occur across animal species from invertebrates to mammals and in all major tissues, serving multiple possible functions (e.g. regulation of actin polymerization, calmodulin-dependent enzyme activity, acyl-CoA metabolism, GABA-A/benzodiazepine receptor modulation, steroidogenesis, etc.). Thus, these data provide the first evidence of specific molecular targets of Pb in kidney of environmentally-exposed humans, and they suggest that low-level Pb toxicity may occur via alteration of T beta 4 and ACBP (DBI) function in renal and other tissues, including the central nervous system.

Lead-binding proteins in brain tissue of environmentally lead-exposed humans.

This study reports the partial purification and characterization of cytosolic lead binding proteins (PbBPs) in human brain tissue of environmentally Pb-exposed subjects. The isolated proteins were initially characterized based upon the presence of endogenously associated Pb. Following partial purification (Sephadex G-75 and A-25 DEAE anion-exchange chromatography), the isolated PbBPs (contained within a single DEAE peak) showed a single class of high affinity binding sites with an apparent Kd of 10(-9) M, based upon competition assays using radioactive 203Pb and Hill and Scatchard analysis. The presence of endogenously bound Pb with the isolated proteins indicated the association of Pb with the protein(s) in vivo in these environmentally Pb-exposed subjects, since the samples were prepared in an ultraclean lead analysis laboratory. Moreover, the persistence of Pb-protein binding throughout the initial two steps (Sephadex G-75 and A-25 DEAE) of the purification scheme is consistent with the high affinity and stability of binding measured with the radiolead competition assays. The DEAE isolated PbBPs were further purified by denaturing reversed-phase HPLC analysis, resulting in the isolation of two proteins, thymosin beta 4 (5 kDa, pI 5.1) and a second as yet unidentified protein with an approximate molecular mass of 20 kDa and a pI of 5.9. Qualitative 203Pb-binding analysis of these HPLC purified proteins suggested that they may be primarily responsible for the observed Pb binding in the single DEAE peak. Nearly identical results were obtained in brain cytosols from male and female, and young and adult individuals, although further quantitative analyses are needed to investigate possible sex and age relationships. These data are significant because they contribute to a better understanding of the presence of PbBPs in a sensitive target organ for Pb toxicity in humans, suggesting a possible role of these or similar proteins as sensitive biomarkers of Pb exposure and toxicity.

Role of lead-binding proteins in renal cancer.

High-dose lead exposure in rodents has been shown to produce pathognomonic lead intranuclear inclusion bodies and to result in an increased incidence of renal adenocarcinomas. Studies from this laboratory and others have demonstrated the presence of high-affinity renal lead-binding proteins in rat kidneys which act as tissue sinks for lead at low dose levels. Cell-free nuclear translocation studies have shown that these molecules are capable of facilitating the intranuclear movement of lead and that they are associated with chromatin. These data suggest that renal lead-binding proteins may play a role in mediating known alterations in renal gene expression associated with formation of intranuclear inclusion bodies. More recent studies from this laboratory have demonstrated the presence of chemically similar lead-binding proteins in kidneys of both monkeys and humans. Such observations suggest that a similar mechanism may be operating in primates since lead intranuclear inclusion bodies are also observed in these species. These data provide a testable mechanistic approach for assessing the possible role(s) of lead-binding proteins in mediating the intranuclear movement of lead and lead-induced renal cancer in primate species.

Detection of DNA point mutations with DNA mismatch repair enzymes.

We have developed a simple and reliable procedure to screen gene mutations using DNA mismatch repair (MR) specific mut Y enzyme of Escherichia coli and thymidine DNA glycosylase from HeLa cells. The mut Y enzyme cleaves A of G/A mismatches in DNA duplex and thymidine glycosylase cleaves T at G/T mismatches. Previously, we showed the determination of G:C-->T:A mutations in the N-ras gene in two human tumor samples with mut Y G/A MR enzyme. As low as 1-2% mutant DNAs in a sample of mutant and wild-type DNA can be detected with a synthetic DNA to create G/A mispairing for the assay. In this paper, we simplify the assay, include G/T MR thymidine glycosylase from HeLa cells and evaluate the application for screening DNA point mutations of p53 and ras genes. In this method, DNA fragments amplified from normal and mutated genes by polymerase chain reaction (PCR) were mixed and annealed to create DNA mismatches for cleavage by mismatch repair enzymes. The cleaved products and the substrates were separated by gel electrophoresis and detected by autoradiography. In theory, the enzymes that cut G/A or G/T mispairs will detect the mutations of G:C-->A:T, A:T-->G:C, G:C-->T:A and T:A-->G:C. Several human tumor samples were examined for p53 or K-ras mutations with G/A and G/T mismatch repair enzymes. The reliability of mutation detection was evaluated by comparing the results with reported mutations or confirmed by DNA sequencing of the same PCR-amplified DNA fragments. Our data showed that, following mismatch repair enzyme cleavage, all mutated DNA samples yielded cleaved products with sizes as expected. In addition, our assay is able to characterize the nature of mutation by 5' end-labeling of 32P on mutant or wild-type DNA fragments. The low background, reliability and the determination of the sites of mutations as well as the types of DNA base changes indicate the advantages of the method over other techniques in testing DNA mutants.

Implications of lead binding proteins for risk assessment of lead exposure.

Lead-binding proteins have previously been isolated from rat and human target tissues. These molecules have shown to possess molecular masses in the general range of 10,000-30,000 daltons. The proteins are acidic in nature and rich in aspartic and glutamic amino acid residues. The molecules in rodents appear to play several important roles in mediating the low dose toxicity of lead in the kidney and brain. Preliminary studies presented in this report indicate that monkeys also possess similar proteins in the kidney and brain, thus providing a biochemical "bridge" in a non-human primate between rodent models and humans. Further, the excretion of these molecules into the urine of rodents increases with lead exposure, suggesting that may also prove useful as biomarkers of lead exposure in humans and monkeys once the dose-range and mechanism(s) of this phenomenon are further defined. Such studies should provide valuable risk assessment information for determining why individuals vary in their susceptibility to lead toxicity.

Methods for improving tissue culture of human tracheo-bronchial epithelium obtained at autopsy.

Human tracheo-bronchial epithelium obtained from autopsy, surgery, and organ donation will have areas of both viable and non-viable cells. It is important in the initial establishment of epithelial explant and cell cultures that injured, non-viable mucosal epithelium not be used for the cultures. Autopsy cases selected for culture should initially be chosen on the basis of a shorter post mortem interval and cause of death in order to increase the rate of successful culture. Staining the epithelium with the vital dye, trypan blue, in combination with phase contrast microscopy of the bronchial tissues will further identify those areas of the mucosa that are enriched for viable cells. The dead, non-viable areas are trypan blue positive, while the viable areas are clear and have foci of beating, motile cilia. Treatment of the mucosal tissue with mucolytic agents to remove cell debris, dead cells, and microbes trapped in the mucus material will further improve the chances for successful culture. Human tracheo-bronchial epithelium, although non-sterile and often injured at time zero for numerous reasons, can effectively be used in vitro pathophysiology studies.

Characterization of an in vitro system of human renal papillary collecting duct cells.

We have developed an in vitro model of human papillary collecting duct cells isolated from cadaver kidneys using methods similar to those we previously reported for the isolation of human proximal tubule cells. To date we have isolated papillary collecting duct cells from 100 normal human kidneys. Papillae were dissected and digested in Cellgro containing 400 U/ml collagenase. Cells were plated on fibronectin-coated culture flasks at a density of 10(4) live cells/ml in Cellgro supplemented with insulin and 10% fetal bovine serum. Confluent monolayers, which were able to withstand 600 mOSM for 8 h, were obtained within 10 to 15 d. Cells of primary isolates and first passages exhibited epithelial cell ultrastructure including cell junctions, microvilli, and cilia. A dark-brown reaction product was observed in these cells when stained by the immunoperoxidase method with peroxidase-labeled peanut lectin (Arachis hypogaea), which binds specifically to human distal tubule and collecting duct cells. These cells were negative for Factor-VIII (a marker for endothelial cells) and gamma-glutamyltransferase (a marker for proximal tubule cells). High activities of the glycolytic enzyme pyruvate kinase and arginine vasopressin-stimulated cAMP production in these cells are consistent with a distal nephron origin. The results indicate that human collecting duct cells can be isolated and cultured to provide an in vitro system to probe pathogenetic mechanisms of potential nephrotoxins.

The effect of testosterone on citrate synthesis and citrate oxidation and a proposed mechanism for regulation of net citrate production in prostate.

Citrate oxidation by rat ventral prostate was reduced by castration and increased by testosterone administration. Similarly, the mitochondrial aconitase activity was decreased by castration; whereas cytosol aconitase was unaffected. The rate of citrate oxidation is extremely low in prostate. Castration also decreased mitochondrial aspartate aminotransferase activity while having no effect on the cytosol isoenzyme. Testosterone markedly stimulated the net production of citrate from aspartate plus glutamate by prostate mitochondria. These studies support the proposal that aspartate is a major source of oxalacetate for citrate production, and that a "glutamate-aspartate-citrate" pathway may be functional in prostate mitochondria. In addition, testosterone can regulate citrate production by a specific effect on mitochondrial aspartate aminotransferase activity. Testosterone also regulates the flux of citrate through the Krebs cycle, but this represents only a small proportion of the citrate accumulated. These conditions would be consistent with the function of prostate epithelium in accumulating and secreting citrate.

Ultrastructural effects of pyridostigmine on neuromuscular junctions in rat diaphragm.

Pyridostigmine bromide, a quaternary carbamate, is widely used in treatment of myasthenia gravis and has been suggested for use in prophylaxis against intoxication with irreversible cholinesterase inhibitors. Since there are virtually no anatomical data concerning the neuromuscular toxicity of the drug, this study was undertaken to evaluate the effects of acute and subacute doses of pyridostigmine on the ultrastrucutre of nerve terminals in rat diaphragm neuromuscular junctions (NMJs). Pyridostigmine in a Mestinon-equivalent buffer was administered by single, subcutaneous injection (acute exposure; 10-30 min) or by a subcutaneously implanted Alzet osmotic minipump (subacute exposure; 2, 7 or 14 days). Acute exposure doses ranged from 0.0036 mg/kg to 3.6 mg/kg (0.001-1.0 LD50), while subacute exposure doses ranged from 0.43 to 20 mg of the drug. Both acute and subacute exposures resulted in dose dependent alterations of presynaptic elements in diaphragmatic NMJs, which included disruption of organelles in the axon terminal, regional or total withdrawal of the nerve terminal from postsynaptic junctional folds, and invasion of Schwann cell fingers into the synaptic cleft. These ultrastructural observations suggest that the normal cell-to-cell interactions at diaphramatic NMJs are altered by this "reversible," cholinesterase inhibiting drug.

Neuromuscular toxicity of pyridostigmine bromide in the diaphragm, extensor digitorum longus, and soleus muscles of the rat.

The neuromuscular junctions from diaphragm, soleus, and extensor digitorum longus (EDL) muscles of male albino rats were assessed for morphological alterations following acute (30-min) and subacute (2-day) exposure to pyridostigmine bromide in Mestinon-equivalent buffer. These muscles were selected to compare the effects of the drug on muscles of different fiber type composition. The diaphragm has approximately equal numbers of type I and type II fibers while the soleus and EDL possess primarily type I and type II fibers, respectively. Pyridostigmine was administered to each acute-exposure animal by a single subcutaneous injection of 0.36 mg/kg pyridostigmine and to each subacute-exposure animal by a subcutaneously implanted osmotic minipump containing 10 mg/ml pyridostigmine. Both treatments resulted in whole blood cholinesterase (ChE) depression of approximately 60-70% as determined by radiometric assay. Control animals received only Mestinon-equivalent buffer. Both acute and subacute exposures resulted in morphological alteration of the neuromuscular junctions (NMJs) of all three muscles, although considerable variation in the extent of damage occurred even within individual NMJs. The most frequently observed presynaptic alterations were mitochondrial damage and partial withdrawal of nerve terminal branches (partial denervation). Post-synaptic changes included occasional rarefaction of mitochondrial matrices and disruption of the myofibrillar organization in small numbers of subjunctional sarcomeres. The data indicate that acute or subacute exposure to pyridostigmine bromide at a whole blood ChE depression of 60-70% results in similar alterations to the NMJs of three muscles with substantially different fiber type compositions. Although the severity of the damage varies from fiber to fiber, the variability appears random and not related to a specific fiber type or dosage regimen.

Mediation of organ specificity in chemical carcinogenesis.

Organ and species specificity in the experimental induction of cancer by chemical agents has been widely observed and is incompletely understood even though the phenomenon has become a persistent puzzle in oncology. Animal model experiments and epidemiological data on human cancer morbidity from environmental causative agents would lead to the conclusion that some organs are more susceptible because they tend to concentrate and (or) retain a given agent more than other tissues. The routes of administration or unique features in the metabolism of the agent explain the differences in some instances only. We propose an additional general mechanism: an organ (or cell type) may be specifically and differentially susceptible to the action of an agent if, by happenstance, a major storage or secretory protein constituent of the cell binds the agent in question reversibly, thereby concentrating or retaining the agent.

Metabolic studies of postischemic acute renal failure in the rat.

Postischemic acute renal failure was induced by 1 hr of clamping of the renal vasculature. Adenine nucleotide (ATP, ADP, AMP) and lactate (Lac) levels were measured after 0, 0.25, 1, 6, 24, and 48 hr of reflow to determine the time necessary for recovery to control levels. After 1 hr of ischemia with no reflow, [ATP] was 18% and [Lac] was 10-fold control levels. Control levels were restored after 24 hr of reflow. Variable ischemic times (5, 15, 30, 60, 90, and 120 min) followed by (1) no reflow or (2) 24 hr of reflow were also studied. [ATP] decreased to 25 and 13% of controls after 5 and 120 min of ischemia, respectively, and [Lac] increased to 5- and 13-fold controls after 5 and 120 min. Five to ninety minutes of ischemia followed by 24 hr of reflow resulted in a trend toward restoration of ATP and Lac levels; whereas, 120 min of ischemia followed by 24 hr of reflow resulted in death. The results indicate that: (1) In vivo ischemia results in a drastic and rapid shift in the ATP-ADP-AMP equilibrium; (2) the absolute concentration of ATP is not a reliable criterion of cell viability, but the ability to resynthesize ATP may be determinant in the reversibility of the lesion; (3) 1 hr of ischemia is reversible with respect to restoration of [ATP] and [Lac], but 24 hr of reflow are needed for restoration; and (4) ischemia for 90 min results in a metabolic derangement which is partially reversible in that metabolite levels are partially restored after 24 hr of reflow. However, 90 min of vascular clamping is not functionally reversible since the majority of animals exhibit severe azotemia and do not survive.

Biochemical and morphological studies on human kidneys preserved for transplantation.

Seven human kidneys that had been preserved for transplantation by pulsatile perfusion were studied to correlate the biochemical data with morphologic changes. Metabolite concentrations in mumol/g wet tissue were ATP = 0.26; ADP = 0.34; AMP = 0.45; lactate = 15.21; pyruvate = 0.23; 3-phosphoglycerate = 0.05; fructose-1,6-bisphosphate = 0.06; and hexose-6-phosphate = 0.03. Enzyme activities in mumol/min . mg protein found in the microsomal fraction were alkaline phosphatase = 0.049 and gamma-glutamyl transpeptidase = 0.844. Morphologically, none of the kidneys showed irreversible cell injury in the renal tubules, but some glomeruli showed areas where the endothelial cells appeared stripped off of the capillary basement membranes, indicating possible perfusion injury. The data suggest that it is the resynthesizing ability, as opposed to the absolute concentration of ATP, which determines the recovery and the subsequent viability of the tissue.

Aryl hydrocarbon hydroxylase in human prostate.

Aryl hydrocarbon hydroxylase (AHH) was present in explant cultures of human prostate obtained from surgery of benign prostatic hyperplasia and was inducible by benz[a]anthracene (BA). The induction of AHH ranged from 14- to 150-fold when compared with control values and 10-fold variation of AHH inducibility among individuals was observed. Epithelial cells grown from human prostate tissue also contained measurable AHH activity and AHH was inducible by BA and 7,12-dimethylbenz[a]anthracene (DMBA). Inducibility of AHH by BA ranged from 2- to 63-fold. The inducibility of AHH by DMBA was always less than that by BA. In cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), there were no changes in AHH activity. These findings support the view that the human prostate is susceptible to environmental polycyclic hydrocarbon carcinogens and that environmental and occupational factors might contribute to the etiology of human prostatic carcinoma.

Aryl hydrocarbon hydroxylase in human bronchial epithelium and blood monocyte.

The inducibility of aryl hydrocarbon hydroxylase (AHH) in human bronchial epithelium and blood monocyte was studied in 11 immediate autopsy patients without lung cancer. When the bronchus was exposed to 10 microgram of benz[a]anthracene (BA)/ml medium in explant culture, the levels of AHH induction in the bronchus were 3- to 29-fold above control levels. The specific enzyme activity ranged from not detectable (i.e., < 0.14) to 2.9 nmol/hour/mg DNA in untreated tissue and from 1.2 to 30 nmol/hour/mg DNA in BA-treated bronchus. The optimum pH for the bronchus AHH was 7.7. Control AHH and BA-induced AHH in bronchus were both inhibited by 100 microM 7,8-benzoflavone in vitro. Induction of AHH in monocytes ranged from 1.5- to 30-fold above that of controls when the cells were exposed to 2 microgram of BA/ml medium in culture. The specific enzyme activity ranged from 1.6 to 19 pmol/hour/10(6) cells in untreated monocytes and from 5.8 to 53 pmol/hour/10(6) cells in BA-treated monocytes. The extent of AHH induction in monocytes depended on BA concentration (from 0.1 to 10.0 microgram) in a dose-related manner. AHH activity increased linearly with the number of monocytes (from 0.5 to 2x10(6)) in the assay system. 7,8-Benzoflavone inhibited the BA-induced but not the basal levels of monocyte AHH activity. The data are consistent with a correlation between the inducibility of AHH in human bronchus and blood monocyte from the same individual.