The Metallothionein-Null Phenotype Is Associated with Heightened Sensitivity to Lead Toxicity and an Inability to Form Inclusion Bodies
2002; Elsevier BV; Volume: 160; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64925-5
ISSN1525-2191
AutoresWei Qu, Bhalchandra A. Diwan, Jie Liu, Robert A. Goyer, Tammy Dawson, John L. Horton, M. George Cherian, Michael P. Waalkes,
Tópico(s)Environmental Toxicology and Ecotoxicology
ResumoSusceptibility to lead toxicity in MT-null mice and cells, lacking the major forms of the metallothionein (MT) gene, was compared to wild-type (WT) mice or cells. Male MT-null and WT mice received lead in the drinking water (0 to 4000 ppm) for 10 to 20 weeks. Lead did not alter body weight in any group. Unlike WT mice, lead-treated MT-null mice showed dose-related nephromegaly. In addition, after lead exposure renal function was significantly diminished in MT-null mice in comparison to WT mice. MT-null mice accumulated less renal lead than WT mice and did not form lead inclusion bodies, which were present in the kidneys of WT mice. In gene array analysis, renal glutathione S-transferases were up-regulated after lead in MT-null mice only. In vitro studies on fibroblast cell lines derived from MT-null and WT mice showed that MT-null cells were much more sensitive to lead cytotoxicity. MT-null cells accumulated less lead and formed no inclusion bodies. The MT-null phenotype seems to preclude lead-induced inclusion body formation and increases lead toxicity at the organ and cellular level despite reducing lead accumulation. This study reveals important roles for MT in chronic lead toxicity, lead accumulation, and inclusion body formation. Susceptibility to lead toxicity in MT-null mice and cells, lacking the major forms of the metallothionein (MT) gene, was compared to wild-type (WT) mice or cells. Male MT-null and WT mice received lead in the drinking water (0 to 4000 ppm) for 10 to 20 weeks. Lead did not alter body weight in any group. Unlike WT mice, lead-treated MT-null mice showed dose-related nephromegaly. In addition, after lead exposure renal function was significantly diminished in MT-null mice in comparison to WT mice. MT-null mice accumulated less renal lead than WT mice and did not form lead inclusion bodies, which were present in the kidneys of WT mice. In gene array analysis, renal glutathione S-transferases were up-regulated after lead in MT-null mice only. In vitro studies on fibroblast cell lines derived from MT-null and WT mice showed that MT-null cells were much more sensitive to lead cytotoxicity. MT-null cells accumulated less lead and formed no inclusion bodies. The MT-null phenotype seems to preclude lead-induced inclusion body formation and increases lead toxicity at the organ and cellular level despite reducing lead accumulation. This study reveals important roles for MT in chronic lead toxicity, lead accumulation, and inclusion body formation. Lead is widely recognized as an important environmental toxicant that poses a substantial risk to the human population throughout the world.1International Agency for Research on Cancer (IARC): IARC Monograph on the Evaluation of Carcinogenic Risks to Humans: Overall Evaluations of Carcinogenicity. An Update of IARC. Lyon, IARC, Monographs 1 to 42, suppl 7, 1987, pp 230–232Google Scholar Toxic effects of lead occur in multiple organ systems but particularly the developing nervous system of infants and children.2Nolan CV Shaikh ZA Lead nephrotoxicity and associated disorders: biochemical mechanisms.Toxicology. 1992; 73: 127-146Crossref PubMed Scopus (154) Google Scholar, 3Goyer RA Lead.in: Bingham E Cohrssen B Powell CH Patty's Toxicology. John Wiley & Sons, New York2001: 611-675Google Scholar Renal effects are also common in adults with chronic lead exposure.3Goyer RA Lead.in: Bingham E Cohrssen B Powell CH Patty's Toxicology. John Wiley & Sons, New York2001: 611-675Google Scholar Lead produces renal tumors in rodents, and lead and inorganic lead compounds have been classified as possible human carcinogens.4International Agency for Research on Cancer (IARC) IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans: Some Metals and Metallic Compounds. IARC, Lyon1980: 325-415Google Scholar However, the precise mechanisms of lead toxicity or carcinogenicity are incompletely defined. A remarkable pathogenic feature of lead poisoning is the presence of inclusion bodies composed of lead-protein complex.5Choie DD Ricchter GW Lead poisoning: rapid formation of intranuclear inclusion.Science. 1972; 177: 1194-1195Crossref PubMed Scopus (82) Google Scholar, 6Blackman SS Intranuclear inclusion-bodies in the kidney and liver caused by lead poisoning.Bull John Hopkins Hosp. 1936; 58: 584Google Scholar, 7Goyer RA May P Cates M Krigman MR Lead and protein content of isolated intranuclear inclusion bodies from kidneys of lead-poisoned rats.Lab Invest. 1970; 22: 245-251PubMed Google Scholar, 8Richter GW Evolution of cytoplasmic fibrillar bodies induced by lead in rat and mouse kidneys.Am J Pathol. 1976; 83: 135-148PubMed Google Scholar, 9Goyer RA Rhyne BC Pathological effects of lead.Int Rev Pathol. 1973; 12: 1-77PubMed Google Scholar, 10Klann E Shelton KR The effect of lead on the metabolism of a nuclear matrix protein which becomes prominent in lead-induced intranuclear inclusion bodies.J Biol Chem. 1989; 264: 16969-16972PubMed Google Scholar, 11Van Mullen PJ Stadhouders AM Bone marking and lead-intoxication: early pathological changes in osteoclasts.Virchows Arch B Cell Pathol. 1974; 15: 345-350PubMed Google Scholar, 12Goyer RA Leonard JF Rhyne B Krigman MR Lead dosage and the role of the intranuclear inclusion body.Arch Environ Health. 1970; 20: 705-711Crossref PubMed Scopus (129) Google Scholar, 13Goyer RA Lead toxicity: a problem in environmental pathology.Am J Pathol. 1971; 64: 167-179PubMed Google Scholar, 14Goyer RA Wilson MH Lead-induced inclusion bodies.Lab Invest. 1975; 32: 149-156PubMed Google Scholar, 15Cherian MG Nordberg M Cellular adaptation in metal toxicology and metallothionein.Toxicology. 1983; 28: 1-15Crossref PubMed Scopus (123) Google Scholar Blackman6Blackman SS Intranuclear inclusion-bodies in the kidney and liver caused by lead poisoning.Bull John Hopkins Hosp. 1936; 58: 584Google Scholar first reported the formation of lead inclusion bodies in the 1930s in renal epithelial cells of lead-poisoned children. Since then, many investigators have reported inclusion body formation with lead exposure in humans and animals.5Choie DD Ricchter GW Lead poisoning: rapid formation of intranuclear inclusion.Science. 1972; 177: 1194-1195Crossref PubMed Scopus (82) Google Scholar, 7Goyer RA May P Cates M Krigman MR Lead and protein content of isolated intranuclear inclusion bodies from kidneys of lead-poisoned rats.Lab Invest. 1970; 22: 245-251PubMed Google Scholar, 8Richter GW Evolution of cytoplasmic fibrillar bodies induced by lead in rat and mouse kidneys.Am J Pathol. 1976; 83: 135-148PubMed Google Scholar Lead-induced inclusion bodies are frequently nuclear, roughly spherical, and typically consist of an electron-dense core with a fibrillary network at the periphery.2Nolan CV Shaikh ZA Lead nephrotoxicity and associated disorders: biochemical mechanisms.Toxicology. 1992; 73: 127-146Crossref PubMed Scopus (154) Google Scholar These inclusion bodies, although common in the kidney, also form in cells of nervous tissue origin such as astrocytes,9Goyer RA Rhyne BC Pathological effects of lead.Int Rev Pathol. 1973; 12: 1-77PubMed Google Scholar neuroblastoma cells,10Klann E Shelton KR The effect of lead on the metabolism of a nuclear matrix protein which becomes prominent in lead-induced intranuclear inclusion bodies.J Biol Chem. 1989; 264: 16969-16972PubMed Google Scholar and in other cell types such as osteoclasts.11Van Mullen PJ Stadhouders AM Bone marking and lead-intoxication: early pathological changes in osteoclasts.Virchows Arch B Cell Pathol. 1974; 15: 345-350PubMed Google Scholar Metal analysis shows that lead is highly concentrated within the inclusion bodies.12Goyer RA Leonard JF Rhyne B Krigman MR Lead dosage and the role of the intranuclear inclusion body.Arch Environ Health. 1970; 20: 705-711Crossref PubMed Scopus (129) Google Scholar Inclusion bodies may be protective in that, when lead accumulates in the inclusion bodies, it prevents injury to more sensitive cellular targets.12Goyer RA Leonard JF Rhyne B Krigman MR Lead dosage and the role of the intranuclear inclusion body.Arch Environ Health. 1970; 20: 705-711Crossref PubMed Scopus (129) Google Scholar, 13Goyer RA Lead toxicity: a problem in environmental pathology.Am J Pathol. 1971; 64: 167-179PubMed Google Scholar It is thought that inclusion bodies probably have an important role in the intracellular partitioning and, perhaps, transport and toxicity of lead.14Goyer RA Wilson MH Lead-induced inclusion bodies.Lab Invest. 1975; 32: 149-156PubMed Google Scholar Thus, the formation of lead-binding inclusion bodies may function to detoxify lead,15Cherian MG Nordberg M Cellular adaptation in metal toxicology and metallothionein.Toxicology. 1983; 28: 1-15Crossref PubMed Scopus (123) Google Scholar although this has yet to be definitively established. Metallothionein (MT) is a low-molecular-weight metal-binding protein with one-third of its amino acids as cysteine.16Waalkes MP Perez-Olle R Role of metallothionein in metabolism, transport, and toxicity of metals.in: Koropatnick DJ Zalups R Molecular Biology and Toxicology of Metals. Taylor & Francis, London2000Google Scholar These cysteinyl sulfhydryls coordinate a variety of metal atoms.17Cherian MG Howell SB Imura N Klaassen CD Koropatnick J Lazo JS Waalkes MP Role of metallothionein in carcinogenesis.Toxicol Appl Pharmacol. 1994; 126: 1-5Crossref PubMed Scopus (43) Google Scholar Various metals increased the concentration of MT in major organs of rats.18Waalkes MP Klaassen CD Concentration of metallothionein in major organs of rats after administration of various metals.Fund Appl Toxicol. 1985; 5: 473-477Crossref PubMed Scopus (121) Google Scholar MT has been assigned pleiotropic roles from gene regulation to metal homeostasis, transport, and detoxification.19Rhee SJ Huang PC Metallothionein accumulation in CHO Cdr cells in response to lead treatment.Chem-Biol Interact. 1989; 72: 347-361Crossref PubMed Scopus (17) Google Scholar For instance, MT has been shown to play a protective role in cadmium-induced hepatotoxicity and nephrotoxicity.20Klaassen CD Liu J Role of metallothionein in cadmium-induced hepatotoxicity and nephrotoxicity.Drug Metab Rev. 1997; 29: 79-102Crossref PubMed Scopus (132) Google Scholar Similarly, MT-I/II knock-out (MT-null) mice are more sensitive than wild-type (WT) mice to the nephrotoxicity produced by chronic exposure to cadmium and/or other inorganic metals.21Liu J Liu Y Habeebu SM Waalkes MP Klaassen CD Chronic combined exposure to cadmium and arsenic exacerbates nephrotoxicity, particularly in metallothionein-I/II null mice.Toxicology. 2000; 147: 157-166Crossref PubMed Scopus (94) Google Scholar MT is highly inducible by many metals, particularly zinc, cadmium, copper, and mercury, and clearly plays a role in mitigating the toxicity of these metals.17Cherian MG Howell SB Imura N Klaassen CD Koropatnick J Lazo JS Waalkes MP Role of metallothionein in carcinogenesis.Toxicol Appl Pharmacol. 1994; 126: 1-5Crossref PubMed Scopus (43) Google Scholar However, any mitigating role for MT in lead toxicity is still only poorly defined. In this regard, lead has been shown to induce the synthesis of MT in several instances,19Rhee SJ Huang PC Metallothionein accumulation in CHO Cdr cells in response to lead treatment.Chem-Biol Interact. 1989; 72: 347-361Crossref PubMed Scopus (17) Google Scholar, 22Maitani T Watahiki A Suzuki KT Induction of metallothionein after lead administration by three injection routes in mice.Toxicol Appl Pharmacol. 1986; 83: 211-217Crossref PubMed Scopus (29) Google Scholar, 23Ikebuchi H Teshima R Suzuki K Terao T Yamane Y Simultaneous induction of lead-metallothionein-like protein and zinc-thionein in the liver of rats given lead acetate.Biochem J. 1986; 233: 541-546Crossref PubMed Scopus (55) Google Scholar, 24Ikebuchi H Teshima R Suzuki K Sawada JI Terao T Yamane Y An immunological study of a lead-thionein-like protein in rat liver.Biochem Biophys Res Commun. 1986; 136: 535-541Crossref PubMed Scopus (15) Google Scholar which implicates, but does not definitively establish, a role in lead metabolism. On the other hand, this induction seems rather modest compared to many other metals and occurs only in the liver,18Waalkes MP Klaassen CD Concentration of metallothionein in major organs of rats after administration of various metals.Fund Appl Toxicol. 1985; 5: 473-477Crossref PubMed Scopus (121) Google Scholar perhaps indicating stress-mediated induction. Others have found that lead is unable to stimulate the synthesis of MT in human blood lymphocytes.25Yamada H Koizumi S Metallothionein induction in human peripheral blood lymphocytes by heavy metals.Chem-Biol Interact. 1991; 78: 347-354Crossref PubMed Scopus (47) Google Scholar Lead appears to bind to MT or MT-like proteins in human erythrocytes,26Church HJ Day JP Braithwaite RA Brown SS Binding of lead to a metallothionein-like protein in human erythrocytes.J Inorg Biochem. 1993; 49: 55-68Crossref PubMed Scopus (47) Google Scholar which suggests sequestration into a nonbioavailable, and thus nontoxic form. The presence of zinc-induced MT will modestly mitigate the toxicity of lead in cultured primary rat hepatocytes27Liu J Kershaw WC Klaassen CD The protective effect of metallothionein on the toxicity of various metals in rat primary hepatocyte culture.Toxicol Appl Pharmacol. 1991; 107: 27-34Crossref PubMed Scopus (100) Google Scholar and lead can avidly bind to MT ex vivo displacing zinc in the process.28Waalkes MP Harvey MJ Klaassen CD Relative in vitro affinity of hepatic metallothionein for metals.Toxicol Lett. 1984; 20: 33-39Crossref PubMed Scopus (145) Google Scholar Furthermore, the binding of lead to MT seems to reduce lead-induced inhibition of the enzyme δ-aminolevulinic acid dehydratase, at least ex vivo.29Goering PL Fowler BA Kidney zinc-thionein regulation of delta-aminolevulinic acid dehydratase inhibition by lead.Arch Biochem Biophys. 1987; 253: 48-55Crossref PubMed Scopus (32) Google Scholar Although there are indications that MT mitigates lead toxicity, the data are far from convincing and additional work is warranted. Therefore, the purpose of the present study was to investigate the role of MT in lead toxicity using genetically engineered systems. Initial studies used MT-null mice that are unable to produce the major forms of MT (MT-I and MT-II isoforms) and compared them to WT controls. Despite accumulating less renal lead, MT-null animals were significantly more sensitive than WT mice to the nephrotoxic effects of lead, as assessed by nephromegaly, renal function, and molecular evidence of a toxic response. Surprisingly, MT-null mice did not form inclusion bodies. Additional work in vitro showed MT-null cells similarly accumulated less lead but were still more sensitive to lead-induced cytotoxicity than WT cells. MT-null cells also did not form inclusion bodies after lead exposure, although they were common in WT cells. These data indicate that MT may play a role in lead toxicity and, possibly, in inclusion body formation. In addition, because the inability to produce MT seems to be related to enhanced susceptibility to lead toxicity, individuals that poorly express MT may have increased susceptibility to lead intoxication. Lead nitrate, lead acetate, and glutamic acid were obtained from Sigma Chemical Company (St. Louis, MO). Nonradioactive cell proliferation assay kit was obtained from Promega (Madison, WI). Homozygous MT-I/II knock-out mice (129-Mt1tm/Bri, Mt2tm/Bri129/SvPCJ background)30Masters BA Kelly EJ Quaife CJ Brinster RL Palmiter RD Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium.Proc Natl Acad Sci USA. 1994; 91: 584-588Crossref PubMed Scopus (581) Google Scholar were obtained from Jackson Laboratories (Bar Harbor, ME). The homozygous mutants were mated inter se to maintain the line. Male MT-null mice and the corresponding WT mice were housed in an American Association for Acreditation of Laboratory Animal Care (AAALAC) accredited facility under conditions that met or exceeded recommendations outlined in the Guide for Care and Use of Laboratory Animals (National Institutes of Health Publication no. 86-23, 1985). Mice were provided food (NIH-31 diet; Zeigler Brothers, Gardners, PA) and water ad libitum. At 10 weeks of age, MT-null and WT mice were randomly divided into three treatment groups of 10 mice each and one control group of 20 mice. They were given acidified drinking water containing lead acetate at concentrations of 1000, 2000, or 4000 ppm lead. Control groups of mice received acidified drinking water. Animals were weighed weekly. Mice were killed after 10 weeks of treatment. Their kidneys were removed and weighed individually. For one-half of the controls (n = 10), and the 1000 and 2000 ppm groups, one kidney was fixed in 10% buffered formalin for histopathological analysis and a portion of the contralateral kidney was frozen in liquid nitrogen and used for subsequent lead determination. For the 2000 ppm group and one-half of the controls (n = 10), half of one kidney was frozen in liquid nitrogen for later RNA isolation. Both kidneys in the 4000-ppm group were used for histopathological analysis including quantitation of inclusion bodies. Formalin-fixed kidneys were embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E) for histological examination. In a separate experiment, urine and orbital blood samples were taken from individual male MT-null and WT mice that were part of an on-going chronic carcinogenesis bioassay and had been exposed to 4000 ppm lead for 20 weeks. Blood urea nitrogen, blood creatinine, and total urinary protein were assessed as biomarkers of renal function and determined through a commercial clinical chemistry laboratory (Ani Lytics, Inc., Gaithersburg, MD). Kidneys removed from WT and MT-null mice were digested in nitric acid (J.T. Baker, Philipsburg, NJ) overnight at 65°C. These digests were used for determination of the renal lead levels by graphite furnace atomic-absorption spectrophotometry with a Perkin-Elmer Model 5000 spectrophotometer. The number of inclusion bodies was counted in three randomly selected H&E-stained kidney sections from each group. In each case a total of 200 randomly selected cells from the inner cortex were scored. The Atlas Mouse 1.2 cDNA expression microarray (1178 genes) was performed according to the manufacturers' instructions. Briefly, 10 to 20 μg of total RNA isolated from MT-null control and lead-treated (2000 ppm) mouse kidneys were converted to [α-32P]-dATP-labeled cDNA probe using MMLV reverse transcriptase and Atlas Mouse Stress CDS primer mix (Clontech, Palo Alto, CA). The 32P-labeled cDNA probe was purified using chroma spin-200 columns, denatured in 0.1 mol/L NaOH, 10 mmol/L ethylenediaminetetraacetic acid at 68°C for 20 minutes, followed by neutralization with an equal volume of 1 mol/L NaH2PO4 for 10 minutes. The membrane was prehybridized with Ultrahyb (Ambion, Austin, TX) for 30 to 60 minutes at 42°C, followed by hybridization overnight at 42°C. Arrays were washed two times in 2× standard saline citrate/0.1% sodium dodecyl sulfate, 5 to 10 minutes each, and two times in 0.1× standard saline citrate/0.1% sodium dodecyl sulfate for 15 to 30 minutes. The arrays were then sealed in a plastic bag, and exposed to a phosphoimage screen or X-ray film. The images were analyzed densitometrically using AtlasImage software. The gene expression intensities were normalized with the sum of eight housekeeping genes on the array (40S ribosomal protein S29, 45-kd calcium-binding protein, β-actin, ornithine decarboxylase, myosin 1-α, G3PDH, hypoxanthine-guanine phosphoribosyltransferase, and phospholipase A2) except for ubiquitin (the hybrid intensity of ubiquitin was saturated). Means and SEM of four hybridizations were calculated for this analysis. A cell line created from the embryonic cells of transgenic mice with a targeted disruption of MT-I/II genes (MT-null cells; also known as MT−/−), along with the corresponding WT control cells (WT; also known as MT+/+) from normal mice, were graciously supplied by Dr. John Lazo, University of Pittsburgh, Pittsburgh, PA. Cells were cultured in Dulbecco's modified Eagle's medium media containing 5% fetal bovine serum as described previously.31Lazo JS Kondo Y Dellapiazza D Michalska AE Choo KH Pitt BR Enhanced sensitivity to oxidative stress in cultured embryonic cells from transgenic mice deficient in metallothionein I and II genes.J Biol Chem. 1995; 270: 5506-5510Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar The precipitation of lead in the medium was controlled by complexing lead nitrate with glutamic acid in equimolar amounts, as detailed in a previous report.32McLachlin JR Goyer RA Cherian MG Formation of lead-induced inclusion bodies in primary rat kidney epithelial cell cultures: effect of actinomycin D and cycloheximide.Toxicol Appl Pharmacol. 1980; 56: 418-431Crossref PubMed Scopus (27) Google Scholar Thus, cells were exposed to lead nitrate (200 μmol/L) with glutamic acid in equimolar amounts for the time specified throughout this study. Promega Cell Titer 96 Nonradioactive Cell Proliferation Assay kits were used to determine acute cytotoxicity of lead in MT-null and WT cells as defined by metabolic integrity. The assay measures the amount of formazan produced by metabolic conversion of Owen's reagent [(3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt; MTS] by dehydrogenase enzymes found in the mitochondria of metabolically active cells. The quantity of formazan product, as measured by absorbance at 490 nm, is directly proportional to the number of living cells. A minimum of 4 replicates of 10,000 cells per well were plated in 96-well plates and allowed to adhere to the plate for 24 hours at which time the media was removed and replaced with media containing various concentrations of lead. Cells were then incubated for an additional 24 hours and cell viability was determined.33Romach EH Zhao CQ Del Razo LM Cebrian ME Waalkes MP Studies on the mechanisms of arsenic-induced self tolerance developed in liver epithelial cells through continuous low-level arsenite exposure.Toxicol Sci. 2000; 54: 500-508Crossref PubMed Scopus (85) Google Scholar LC50 values were determined from analysis of the linear portion of the metabolic integrity curves and compared between WT and MT-null cells. WT and MT-null cells were treated with lead (200 μmol/L) for 48 hours. The cells were harvested by trypsinization and fixed overnight in 3% glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.3. After primary fixation, the cells were rinsed in 0.1 mol/L of phosphate buffer for 15 minutes. Postfixation was done in 1% osmium tetroxide in 0.1 mol/L of phosphate buffer, pH 7.3, for 2 hours. The cells were rinsed again in the phosphate buffer for 15 minutes and were followed by treatment with an aqueous solution of 5% uranyl acetate for 2 hours. After dehydration in graded ethanol, the specimens were embedded in PolyBed resin. The resin blocks were cut at ∼90 nm, collected on coated grids, and stained with uranyl acetate and lead citrate. The examination of the grids was done using a Philips 400 electron microscope.32McLachlin JR Goyer RA Cherian MG Formation of lead-induced inclusion bodies in primary rat kidney epithelial cell cultures: effect of actinomycin D and cycloheximide.Toxicol Appl Pharmacol. 1980; 56: 418-431Crossref PubMed Scopus (27) Google Scholar WT and MT-null cells were grown to ∼50% confluence, then the medium was removed and replaced with either fresh control medium or medium containing lead (200 μmol/L). Cells were harvested 24 hours later, counted, and pelleted by centrifugation. The cell pellets were digested overnight in 50% perchloric:nitric acid (2:1). These digests were used for determination of the amount of total lead that had accumulated after 24 hours of exposure. To estimate lead efflux, replicate sets of cells were washed after 24 hours of exposure to lead and allowed to incubate an additional 24 hours in fresh media. These cells were then digested and analyzed for lead. Total cellular lead levels were determined by graphite furnace atomic-absorption spectrophotometry using a Perkin-Elmer Model 5000 spectrophotometer and adjusted to cell numbers. Triplicate determinations were used for each data point. WT cells were treated with lead (200 μmol/L) for 48 hours. Cells were harvested by trypsinization and resuspended at a density of 2.5 × 106/ml in 10 mmol/L Tris buffer (pH 7.4) at 4°C. Cells were then lysed by sonication on ice. Complete lysis was confirmed microscopically and cellular debris was removed by centrifugation (15 minutes, 16,000 × g). MT levels were determined in the supernatant using the Cd-hemoglobin method of Onosaka and colleagues34Onosaka S Tanaka K Doi M Okahara K A simplified procedure for determination of metallothionein in animal tissues.Eisei Kagaku. 1978; 24: 128-131Crossref Scopus (233) Google Scholar as modified by Eaton and Toal.35Eaton DL Toal BF Evaluation of the Cd/hemoglobin affinity assay for the rapid determination of metallothionein in biological tissues.Toxicol Appl Pharmacol. 1982; 66: 134-142Crossref PubMed Scopus (489) Google Scholar Student's t-test or analysis of variance with subsequent Dunnett's test were used as appropriate. All values are expressed as mean ± SEM of three or more replications. Differences were considered significant at level of P < 0.05. Male MT-null and WT mice received lead in drinking water (0 to 4000 ppm; 10 to 20 weeks) and renal pathology and function were assessed. Lead did not alter body weight in either MT-null or WT mice throughout the exposure period (data not shown). MT-null mice showed a dose-related nephromegaly indicative of renal toxicity, although the kidneys of WT mice were unaffected by lead (Figure 1). Although, no microscopically obvious pathological lesions occurred in lead-exposed kidneys, after chronic exposure to 4000 ppm lead in the drinking water MT-null mice showed evidence of diminished renal function when compared to WT mice. Specifically, there were significant (P ≤ 0.05) increases in blood creatinine (0.53 ± 0.03 mg/dl; mean ± SEM, n = 3 to 4) and total urinary protein (206 ± 14.1 mg/dl) in lead-treated MT-null mice when compared to similarly treated WT mice (blood creatinine = 0.33 ± 0.03 mg/dl; total urinary protein = 158 ± 1.76 mg/dl). Additionally, increases in blood urea nitrogen occurred in lead-exposed MT-null mice (30.3 ± 0.32 mg/dl) that approached significance (P = 0.062) when compared to WT mice (26.7 ± 1.86 mg/dl). This pattern of increases in blood urea nitrogen, blood creatinine, and total urinary protein is typically considered functional evidence of nephrotoxicity, and is consistent with reports on lead-induced nephrotoxicity. Surprisingly, MT-null mice did not form renal lead-containing inclusion bodies, whereas inclusion bodies were common at all doses in WT mice (Figure 2). These inclusion bodies were primarily nuclear. Quantitative analysis of cells from the inner cortex of lead-treated and control sections of kidneys showed that inclusion bodies were increased in a dose-dependent manner in WT mice, but, again were completely absent from MT-null animals (Table 1).Table 1Quantitation of Lead-Induced Inclusion Body Formation in Kidney From WT MiceLead dose (ppm, p.o.)Mouse strain0100020004000WTN.D.10± 116± 121± 1MT-nullN.D.N.D.N.D.N.D.WT and MT-null mice were given lead p.o. at 0, 1000, 2000, or 4000 ppm for 10 weeks and renal inclusion body formation was assessed. In each case 200 nuclei selected from random fields of the inner cortex of lead-treated and control sections of kidneys were scored. Data are given as the mean ± SEM (n = 3).N.D., not detected. Open table in a new tab WT and MT-null mice were given lead p.o. at 0, 1000, 2000, or 4000 ppm for 10 weeks and renal inclusion body formation was assessed. In each case 200 nuclei selected from random fields of the inner cortex of lead-treated and control sections of kidneys were scored. Data are given as the mean ± SEM (n = 3). N.D., not detected. Lead-treated WT kidneys were analyzed immunohistochemically for MT localization to see if MT played a direct role in lead-induced inclusion body formation. MT in lead-treated kidneys from WT mice was primarily cytosolic with minimal nuclear staining and no apparent association with inclusion bodies (data not shown). After 10 weeks of exposure to 0, 1000, or 2000 ppm lead in drinking water, renal lead levels were determined in MT-null and WT mice. Surprisingly, MT-null mice accumulated significantly less renal lead than WT mice at all doses tested (Table 2).Table 2Lead Accumulation in Kidney from WT and MT-Null Mice (μg/g Wet Weight)Lead dose (ppm, p.o.)Mouse strain010002000WTN.D.10.9 ± 0.314.4 ± 0.3MT-nullN.D.8.9 ± 0.2*Significant (P < 0.05) difference from appropriate dose-matched WT mice.11.0 ± 0.7*Significant (P < 0.05) difference from appropriate dose-matched WT mice.WT and MT-null mice were given lead p.o. at 0, 1000, or 2000 ppm for 10 weeks, renal lead levels were measured by AAS. Data given as the mean ± SEM (n = 10).N.D., not detectable.* Significant (P < 0.05) difference from appropriate dose-matched WT mice. Open table in a new tab WT and MT-null mice were given lead p.o. at 0, 1000, or 2000 ppm for 10 weeks, renal lead levels were measured by AAS. Data given as the mean ± SEM (n = 10). N.D., not detectable. To help define more subtle differential toxicity after lead exposure, gene expression array studies were performed with RNA isolated from the kidneys of lead-treated (2000 ppm for 10
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