RESUMO
Nanoparticles can be formed following degradation of medical devices such as orthopedic implants. To evaluate the safety of titanium alloy orthopedic materials, data are needed on the long-term distribution and tissue effects of injected titanium nanoparticles in experimental animals. In this study, we evaluated the tissue distribution and histopathological effects of titanium dioxide (TiO(2)) nanoparticles (approximately 120 nm diameter) in mice after intravenous (i.v.; 56 or 560 mg kg(-1) per mouse) or subcutaneous (s.c.; 560 or 5600 mg kg(-1) per mouse) injection on two consecutive days. Animals were examined 1 and 3 days, and 2, 4, 12 and 26 weeks after the final injection. When examined by light microscopy, particle agglomerates identified as TiO(2) were observed mainly in the major filtration organs - liver, lung and spleen - following i.v. injection. Particles were still observed 26 weeks after injection, indicating that tissue clearance is limited. In addition, redistribution within the histological micro-compartments of organs, especially in the spleen, was noted. Following s.c. injection, the largest particle agglomerates were found mainly in the draining inguinal lymph node, and to a lesser extent, the liver, spleen and lung. With the exception of a foreign body response at the site of s.c. injection and the appearance of an increased number of macrophages in the lung and liver, there was no histopathological evidence of tissue damage observed in any tissue at any time point.
Assuntos
Fígado/metabolismo , Pulmão/metabolismo , Linfonodos/metabolismo , Nanopartículas , Titânio , Animais , Feminino , Injeções Intravenosas , Injeções Subcutâneas , Fígado/patologia , Pulmão/patologia , Linfonodos/patologia , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/toxicidade , Baço/metabolismo , Baço/patologia , Fatores de Tempo , Distribuição Tecidual , Titânio/administração & dosagem , Titânio/metabolismo , Titânio/toxicidadeRESUMO
Limited experimental models exist to assess drug toxicity in pediatric populations. We recently reported how a multi-age rat model could be used for pre-clinical studies of comparative drug toxicity in pediatric populations. The objective of this study was to expand the utility of this animal model, which previously demonstrated an age-dependent sensitivity to the classic nephrotoxic compound, gentamicin, to another nephrotoxicant, namely cisplatin (Cis). Sprague-Dawley rats (10, 25, 40 and 80 days old) were injected with a single dose of Cis (0, 1, 3 or 6 mg kg(-1) i.p.). Urine samples were collected prior and up to 72 h after treatment in animals that were >or= 25 days old. Several serum, urinary and 'omic' injury biomarkers as well as renal histopathology lesions were evaluated. Statistically significant changes were noted with different injury biomarkers in different age groups. The order of age-related Cis-induced nephrotoxicity was different than our previous study with gentamicin: 80 > 40 > 10 > 25 day-old vs 10 >or= 80 > 40 > 25-day-old rats, respectively. The increased levels of kidney injury molecule-1 (Kim-1: urinary protein/tissue mRNA) provided evidence of early Cis-induced nephrotoxicity in the most sensitive age group (80 days old). Levels of Kim-1 tissue mRNA and urinary protein were significantly correlated to each other and to the severity of renal histopathology lesions. These data indicate that the multi-age rat model can be used to demonstrate different age-related sensitivities to renal injury using mechanistically distinct nephrotoxicants, which is reflected in measurements of a variety of metabolite, gene transcript and protein biomarkers.
Assuntos
Envelhecimento/fisiologia , Cisplatino/toxicidade , Nefropatias/induzido quimicamente , Rim/metabolismo , Fatores Etários , Animais , Biomarcadores/metabolismo , Biomarcadores/urina , Criança , Suscetibilidade a Doenças/metabolismo , Suscetibilidade a Doenças/patologia , Gentamicinas/toxicidade , Humanos , Rim/patologia , Nefropatias/patologia , Nefropatias/urina , Modelos Animais , Pediatria , Ratos , Ratos Sprague-Dawley , Sensibilidade e EspecificidadeRESUMO
Pretreatment with Mn2+ is known to produce tolerance to Cd2+-induced lethality. This study was designed to determine the mechanism of tolerance to Cd2+-induced lethality and hepatotoxicity following Mn2+ pretreatment. Rats given 36 mumoles Cd2+/kg, i.v., died within 10-20 hr while only one of nine rats pretreated with Mn2+ (250 mumoles/kg, s.c., 48 and 24 hr prior to Cd2+ challenge) died. Ten hours after Cd2+, plasma aspartate aminotransferase and sorbitol dehydrogenase activities were elevated markedly, and extensive histopathologic lesions of the liver were evident in control rats but not in Mn2+-pretreated rats. To examine the mechanism of this tolerance, distribution of Cd2+ to fourteen organs and the subcellular distribution in six organs were determined in control and Mn2+-pretreated rats. Two hours after challenge (31 mumoles Cd2+/kg, i.v., 0.75 microCi 109Cd2+/mumol Cd2+), the distribution of Cd2+ to liver markedly increased after Mn2+ pretreatment with concomitant decreases in other tissues. Mn2+ pretreatment also resulted in a marked difference in the hepatic subcellular distribution of Cd2+ with more present in cytosol and less associated with organelles. Gel-filtration chromatography indicated that most cytosolic Cd2+ was bound to a low molecular weight protein. Isolation and partial characterization of this protein suggest that it is identical to metallothionein (MT); it had a similar relative elution following gel-filtration chromatography, had low absorbance at 280 nm and, after separation into two isoproteins by DEAE A-25 anion exchange chromatography, had the same mobility after electrophoresis on non-denaturing polyacrylamide gels as Cd2+-induced metallothioneins. These data suggest that Mn2+ pretreatment reduces Cd2+-induced hepatotoxicity by altering the hepatic subcellular distribution of Cd2+ with more Cd2+ binding to MT in the cytosol. This decreased hepatotoxicity is probably responsible for tolerance to Cd2+-induced lethality.
Assuntos
Cádmio/toxicidade , Fígado/efeitos dos fármacos , Manganês/farmacologia , Animais , Cádmio/metabolismo , Tolerância a Medicamentos , Fígado/metabolismo , Fígado/patologia , Masculino , Metalotioneína/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The present series of studies demonstrate that several high-affinity metal-binding proteins regulate essential metal availability and play a role in metal detoxication. Rat kidney PbBP and ZnMT were shown to mediate both the interaction of Zn and Pb to a Zn metalloenzyme, that is, ALAD. The mechanism for this interaction involves the donation of Zn from PbBP and ZnMT to ALAD and chelation of Pb to these proteins. In addition to activating ALAD via donation of Zn from ZnMT to the enzyme, kidney apothionein was shown to deactivate ALAD. Thus, these proteins may provide an intracellular reservoir for essential metals such as Zn and Cu, serving as a homeostatic control mechanism to readily dispense and sequester these cations to meet cellular metabolic requirements, such as the regulation of metalloenzymes, including ALAD. Although Pb does not effectively induce the synthesis of PbBP or ZnMT, these proteins, via binding of Pb, alter the biological activity of Pb toward a highly sensitive target molecule for Pb, that is, ALAD. Finally, when ALAD is utilized as a biological indicator for Pb exposure, the effect of ZnMT on Pb availability and ALAD activity must be taken into consideration. Tissue-specific differences in these high-affinity, metal-binding proteins and Zn status may alter the expected relationships between total tissue Pb concentrations and inhibition of ALAD.
Assuntos
Proteínas de Transporte/metabolismo , Chumbo/metabolismo , Metalotioneína/metabolismo , Sintase do Porfobilinogênio/metabolismo , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Bovinos , Cobre/metabolismo , Rim/efeitos dos fármacos , Rim/enzimologia , Chumbo/toxicidade , Fígado/efeitos dos fármacos , Fígado/enzimologia , Ratos , Zinco/metabolismoRESUMO
Bisphenol A (BPA) exhibits many estrogen-like effects in the rodent uterus, but not all of these can be attenuated by antiestrogens. This suggests the involvement of alternate pathways of BPA action that do not involve the estrogen receptor (ER). An examination of the in vivo effects of BPA on uterine gene expression and protein levels should contribute to an understanding of its mechanism of action. In this study we examined the dose-related effects of BPA on levels of a suite of heat shock proteins (hsps) and on the localization of hsp90alpha, a chaperone of the ER, in uteri of ovariectomized B6C3F1 mice and compared these effects with those of beta-estradiol (E2). The antiestrogen ICI 182,780 (ICI) was co-administered with BPA or E2 in order to examine the potential role of the ER. BPA, although less potent than E2, increased hsp90alpha and grp94 to similar levels, but was much less effective than E2 in increasing levels of hsp72. Treatment with 100 mg BPA/kg/day or 2 microg E2/kg/day increased hsp90alpha to 300% of control levels and altered its tissue expression pattern. In uteri of corn oil (control)-treated mice, hsp90alpha predominantly localized in the cytoplasm and nuclei of epithelial cells. Upon treatment with BPA or E2 there was increased intensity of staining in the stroma and myometrium, and in the epithelium hsp90alpha was localized almost exclusively in the cytoplasm. The effects of BPA or E2 on hsp levels and hsp90alpha localization were attenuated by ICI. These results suggest an involvement of the ER in BPA- and E2-induced increases in uterine levels of hsp90alpha, grp94, and hsp72, and localization of hsp90alpha.
Assuntos
Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Fenóis/farmacologia , Útero/efeitos dos fármacos , Animais , Compostos Benzidrílicos , Western Blotting , Citoplasma/efeitos dos fármacos , Relação Dose-Resposta a Droga , Estradiol/administração & dosagem , Antagonistas de Estrogênios/administração & dosagem , Estrogênios não Esteroides/administração & dosagem , Estrogênios não Esteroides/farmacologia , Feminino , Fulvestranto , Imuno-Histoquímica , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos , Miométrio/efeitos dos fármacos , Ovariectomia , Fenóis/administração & dosagem , Receptores de Estrogênio/fisiologia , Fatores de Tempo , Útero/metabolismoRESUMO
The ability of the environmental xenoestrogen bisphenol A (BPA) to increase uterine wet weight in the rodent remains controversial, and few studies have previously examined the effects of BPA on uterine morphology. Furthermore, it is not known whether BPA-induced uterotrophic effects are, similarly to beta-estradiol (E(2)), mediated through the estrogen receptor (ER). In this study, we compared the effects of BPA on uterine wet weight and morphology to those of E(2) in the B6C3F1 ovariectomized mouse. To examine whether these effects were mediated through the ER, the antiestrogen ICI 182, 780 (ICI) was co-administered with BPA or E(2). We report that subcutaneous administration of BPA at doses between 0.8 and 8 mg/day over 4 days significantly increased mean uterine wet weights above those of vehicle (corn oil)-treated mice. The uterine weight data suggest that BPA acts as a partial agonist with an EC(50) of 0.72 mg/day compared to 19.4 ng/day for E(2). BPA (2 mg/day) and E(2) (40 ng/day) induced a significant increase in luminal epithelial height and in the thickness of both the stromal and myometrial layers of the uterus. The effects of 40 ng E(2)/day on all endpoints studied were reversed by 20 microg ICI/day. ICI at 200, but not 20 microg/day, was able to reverse the BPA (2 mg/day)-induced increase in both uterine wet weight and luminal epithelial height. ICI alone at 200 microg/day stimulated an increase in thickness of both the stroma and myometrium and did not reverse the effects of BPA (2 mg/day) on these layers. These results suggest that the BPA-induced increase in uterine wet weight and in luminal epithelial height in the ovariectomized B6C3F1 mouse are mediated by the ER.
Assuntos
Estrogênios não Esteroides/toxicidade , Fenóis/toxicidade , Receptores de Estrogênio/efeitos dos fármacos , Útero/efeitos dos fármacos , Animais , Compostos Benzidrílicos , Relação Dose-Resposta a Droga , Feminino , Camundongos , Tamanho do Órgão/efeitos dos fármacos , Ovariectomia , Receptores de Estrogênio/fisiologia , Útero/patologiaRESUMO
Cells respond to physiologic stress by enhancing the expression of specific stress proteins. Heat-shock proteins (hsps) and glucose-regulated proteins (grps) are members of a large superfamily of proteins collectively referred to as stress proteins. This particular stress-protein response has evolved as a cellular strategy to protect, repair, and chaperone other essential cellular proteins. The objective of this study was to evaluate the differential expression of four hsps in the renal cortex and medulla during experimental nephrotoxic injury using HgCl2. Male Sprague-Dawley rats received single injections of HgCl2 (0.25, 0.5, or 1 mg Hg/kg, i.v.). At 4, 8, 16, or 24 h after exposure, kidneys were removed and processed for histopathologic, immunoblot, and immunohistochemical analyses. Nephrosis was characterized as minimal or mild (cytoplasmic condensation, tubular epithelial degeneration, single cell necrosis) at the lower exposures, and progressed to moderate or severe (nuclear pyknosis, necrotic foci, sloughing of the epithelial casts into tubular lumens) at the highest exposures. Western blots of renal proteins were probed with monoclonal antibodies specific for 4 hsps. In whole kidney, Hg(II) induced a time- and dose-related accumulation of hsp72 and grp94. Accumulation of hsp72 was predominantly localized in the cortex and not medulla, while grp94 accumulated primarily in the medulla but not cortex. The high, constitutive expression of hsp73 did not change as a result of Hg(II) exposure, and it was equally localized in cortex and medulla. Hsp90 was not detected in kidneys of control or Hg-treated rats. Since hsp72 has been shown involved in cellular repair and recovery, and since Hg(II) damage occurs primarily in cortex, we investigated the cell-specific expression of this hsp. Hsp72 accumulated primarily in undamaged distal convoluted tubule epithelia, with less accumulation in undamaged proximal convoluted-tubule epithelia. These results demonstrate that expression of specific stress proteins in rat kidney exhibits regional heterogeneity in response to Hg(II) exposure, and a positive correlation exists between accumulation of some stress proteins and acute renal cell injury. While the role of accumulation of hsps and other stress proteins in vivo prior to or concurrent with nephrotoxicity remains to be completely understood, these stress proteins may be part of a cellular defense response to nephrotoxicants. Conversely, renal tubular epithelial cells that do not or are unable to express stress proteins, such as hsp72, may be more susceptible to nephrotoxicity.
Assuntos
Proteínas de Choque Térmico/biossíntese , Córtex Renal/efeitos dos fármacos , Medula Renal/efeitos dos fármacos , Cloreto de Mercúrio/toxicidade , Nefrose/induzido quimicamente , Animais , Especificidade de Anticorpos , Proteínas de Transporte/biossíntese , Eletroforese em Gel de Poliacrilamida , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP72 , Proteínas de Choque Térmico HSP90/biossíntese , Córtex Renal/metabolismo , Córtex Renal/patologia , Medula Renal/metabolismo , Medula Renal/patologia , Masculino , Proteínas de Membrana/biossíntese , Nefrose/metabolismo , Nefrose/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Inorganic arsenic is considered a high-priority hazard, particularly because of its potential to be a human carcinogen. In exposed human populations, arsenic is associated with tumors of the lung, skin, bladder, and liver. While it is known to be a human carcinogen, carcinogenesis in laboratory animals by this metalloid has never been convincingly demonstrated. Therefore, no animal models exist for studying molecular mechanisms of arsenic carcinogenesis. The apparent human sensitivity, combined with our incomplete understanding about mechanisms of carcinogenic action, create important public health concerns and challenges in risk assessment, which could be met by understanding the role of metabolism in arsenic toxicity and carcinogenesis. This symposium summary covers three critical major areas involving arsenic metabolism: its biodiversity, the role of arsenic metabolism in molecular mechanisms of carcinogenesis, and the impact of arsenic metabolism on human risk assessment. In mammals, arsenic is metabolized to mono- and dimethylated species by methyltransferase enzymes in reactions that require S-adenosyl-methionine (SAM) as the methyl donating cofactor. A remarkable species diversity in arsenic methyltransferase activity may account for the wide variability in sensitivity of humans and animals to arsenic toxicity. Arsenic interferes with DNA methyltransferases, resulting in inactivation of tumor suppressor genes through DNA hypermethylation. Other studies suggest that arsenic-induced malignant transformation is linked to DNA hypomethylation subsequent to depletion of SAM, which results in aberrant gene activation, including oncogenes. Urinary profiles of arsenic metabolites may be a valuable tool for assessing human susceptibility to arsenic carcinogenesis. While controversial, the idea that unique arsenic metabolic properties may explain the apparent non-linear threshold response for arsenic carcinogenesis in humans. In order to address these outstanding issues, further efforts are required to identify an appropriate animal model to elucidate carcinogenic mechanisms of action, and to define dose-response relationships.
Assuntos
Arsênio/metabolismo , Arsênio/toxicidade , Carcinógenos/toxicidade , Metiltransferases/metabolismo , Animais , Humanos , Oncogenes , Fenótipo , Medição de Risco , Especificidade da EspécieRESUMO
Biomarkers are important tools which enable toxicologists to reliably predict and detect exposures to xenobiotics and resultant cell injury, ultimately improving risk assessments. Since the de novo synthesis of stress proteins can be detected early after exposure to some agents, analysis of toxicant-induced changes in gene expression, i.e. alterations in patterns of protein synthesis, may be useful to develop as biomarkers of exposure and toxicity. We are utilizing various xenobiotics as tools to study stress protein synthesis in target organs in order to evaluate the target tissue-specificity of this response. Previous data from this laboratory have demonstrated that induction of stress proteins in rat liver, but not kidney, after acute exposure to CdCl2 precedes hepatoxicity. Since kidney is a target tissue after chronic Cd exposure, it was of interest to examine stress protein synthesis in this tissue. However, dose-limiting hepatotoxicity precluded this evaluation. Cd complexed with molecules such as cysteine (cys) or metallothionein has been used in acute dosing regimens as a tool in order to study the nephrotoxicity of Cd. Therefore, this study was undertaken in order to evaluate Cd-induced stress protein synthesis in an important tissue known to be injured after chronic exposure, i.e. kidney. Specific objectives included comparing stress protein synthesis in rat kidney and liver after acute exposure to Cd-cys and CdCl2, determining the Cd threshold concentration for renal stress protein synthesis and assessing the relationship between stress protein synthesis and nephropathy. Male rats were exposed to equivalent doses of Cd as CdCl2 or Cd-cysteine (molar ratio Cd:cys = 1:15). Kidney Cd concentrations increased 5-fold after i.v. injection of Cd-cys compared to CdCl2, mimicking Cd distribution following chronic exposure. After exposure to Cd, tissue slices were incubated with 35S-methionine. Slices were subsequently homogenized and centrifuged, and the 16,000 g supernatants were subjected to SDS-polyacrylamide gel electrophoresis. Proteins which had incorporated 35S-methionine were detected by autoradiography. De novo synthesis of 70, 90 and 110 kDa proteins was enhanced in liver, but not in kidney, 4 h after injection of 2 mg Cd/kg as CdCl2. In contrast, dose-related increases in synthesis of these proteins were observed in kidney 4 h after injection of 1 and 2mg Cd/kg as Cd-cys, but not at lower dosages. In addition, synthesis of a 68 kDa kidney protein was inhibited at 2 mg Cd/kg as Cd-cys. The threshold for Cd-induced stress protein synthesis was shown to be between 4 and 8 micrograms Cd/g tissue.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Cádmio/toxicidade , Proteínas de Choque Térmico/biossíntese , Rim/efeitos dos fármacos , Rim/metabolismo , Animais , Biomarcadores/análise , Cádmio/administração & dosagem , Cádmio/metabolismo , Cloreto de Cádmio , Cloretos/administração & dosagem , Cloretos/metabolismo , Cloretos/toxicidade , Cisteína , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Proteínas de Choque Térmico/análise , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
The interaction of lead (Pb) with proteins may represent a fundamental mechanism by which Pb exerts toxicity. In this overview, various factors which influence the interaction of Pb with proteins will be discussed. Pb interacts with enzyme functional groups, and high-affinity metal-binding proteins, such as Pb-binding proteins and metallothioneins, can mediate this Pb-enzyme interaction. Many other factors influence Pb-protein interactions including ligand competition and binding affinities; protein folding and the nature of the metal-binding site; rates of protein synthesis and degradation; and intracellular localization of the ligand and metal. The remainder of this overview will focus on specific examples of important proteins known to be influenced by Pb or which hypothetically may be influenced by Pb. Gaps in knowledge and important research needs are emphasized. Many of the factors discussed play a role in the relative sensitivity of various enzymes in heme biosynthesis to Pb. Disruption of this critical pathway by Pb may result in neuropathologies and accumulation of neurotoxic heme precursors. High-affinity metal-binding proteins have been shown to play a role in mediating Pb inhibition of the octameric Zn-containing enzyme, ALA dehydratase. Knowledge of regional localization in brain and the postnatal ontogeny of the high-affinity metal-binding proteins may be pivotal in understanding Pb neurotoxicity. Other specific examples related to or potentially related to Pb toxicity which are discussed include nucleic acid binding proteins, calmodulin, protein kinase C, and carbonic anhydrase. These proteins will serve as models to understand some basic principles and differences in Pb-protein interactions.
Assuntos
Proteínas de Transporte/metabolismo , Intoxicação por Chumbo/metabolismo , Chumbo/metabolismo , Animais , Anidrases Carbônicas/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Heme/biossíntese , Humanos , Ligação Proteica , Transdução de Sinais/efeitos dos fármacosRESUMO
Apoptosis is a mode of cell death with morphologic and biochemical features that distinguish it from necrosis. Recent studies demonstrating that mercury compounds initiate apoptosis in cultured cells did not elucidate if the biochemical mechanism of apoptosis involved a dependence on macromolecular synthesis post-insult, i.e. programmed cell death. The objectives of this in vitro study were (1) to determine if HgCl2 cytotoxicity includes an apoptotic component, and (2) to determine if apoptosis is dependent on protein synthesis, i.e. proceeds by an inducible mechanism. Suspensions of mouse lymphoma (L5178Y-R) cells were exposed to 0, 1, 5, or 10 microM HgCl2 and apoptosis was evaluated utilizing qualitative and quantitative methods. At 24 h after exposure, transmission electron microscopy revealed a concentration-related increase in morphologic changes typical of apoptosis: margination of condensed chromatin to the nuclear membrane, dilation of the rough endoplasmic reticulum, cytoplasmic condensation and vacuolation, nuclear dissolution, and plasma membrane blebbing. An increase in Hg-induced DNA fragmentation (DNA 'ladder') was observed using agarose gel electrophoresis. Time- and concentration-dependent increases in the percent of apoptotic cells were observed at 1, 6, 12, and 24 h after HgCl2 exposure using a flow cytometric method that discriminates between cells according to size and granularity. Pretreatment of cells with cycloheximide (CHX), an inhibitor of translation, prior to HgCl2 exposure resulted in a 25-50% reduction in apoptotic cells 24 h after exposure to 10 and 20 microM HgCl2, and concomitantly reduced the overall cytotoxicity compared to HgCl2 alone. These results, although limited to a single cell line, support the hypothesis that HgCl2 induces apoptosis that is dependent, at least in part, upon protein synthesis.
Assuntos
Apoptose/efeitos dos fármacos , Cloreto de Mercúrio/toxicidade , Proteínas/efeitos dos fármacos , Animais , Apoptose/genética , Sobrevivência Celular/efeitos dos fármacos , Cicloeximida/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Biossíntese de Proteínas , Inibidores da Síntese de Proteínas/farmacologia , Fatores de Tempo , Células Tumorais Cultivadas/citologia , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/ultraestruturaRESUMO
Humans risk inadvertent intraperitoneal or intravenous exposure to formaldehyde (HCHO), commonly used for disinfection of implanted or extracorporeal medical devices. Various chemical and physical stresses are known to induce hepatic metallothionein. This study examined the effect of acute parenteral administration of HCHO on induction of hepatic metallothionein synthesis. Adult male CF1 mice were administered HCHO ip and hepatic metallothionein was quantified by the cadmium-radioassay method. HCHO (50 mg/kg) increased hepatic metallothionein as early as 8 hr after dosing with maximal levels (27-fold increase) occurring at 72 hr. Metallothionein concentrations were elevated (15-fold) 24 hr after 50 or 100 mg HCHO/kg but not at lower dosages. Concomitant elevations in hepatic zinc and copper content were observed. No increases in metallothionein were observed in kidney, pancreas, or intestine 24 hr after HCHO administration (100 mg/kg, ip). Induction of metallothionein by HCHO may reflect direct de novo synthesis since the response was abolished by pretreatment with the RNA synthesis inhibitor, actinomycin D. HCHO induction of metallothionein also does not appear to be mediated by stress-induced release of corticosteroids or catecholamines from the adrenal since the response was unaltered in adrenalectomized mice. Interference with the glutathione (GSH)-dependent oxidation of HCHO by reducing hepatic GSH concentrations to 40% of control after a 2-hr pretreatment with phorone decreased the metallothionein induction response to HCHO by 33%. This result suggests that the induction may be partially due to a HCHO metabolite, e.g., formate. Confirmation of metallothionein synthesis was obtained following spectral and chromatographic analysis. Thus, HCHO and/or a metabolite produces a marked increase in hepatic metallothionein and alters hepatic zinc and copper homeostasis, all of which are transient responses. Although HCHO was only mildly hepatotoxic at the highest dose (as evidenced by an increase in plasma alanine aminotransferase activity), such changes in metallothionein synthesis and essential metal homeostasis may be part of a cellular repair mechanism operant after acute toxic cell injury.
Assuntos
Formaldeído/toxicidade , Fígado/efeitos dos fármacos , Metalotioneína/biossíntese , Animais , Cobre/análise , Formaldeído/metabolismo , Glucocorticoides/fisiologia , Glutationa/metabolismo , Cetonas/farmacologia , Fígado/metabolismo , Masculino , Camundongos , Biossíntese de Proteínas , Zinco/análiseRESUMO
This study was undertaken to evaluate the ability of kidney Zn-thionein to regulate Zn availability to the Zn-dependent enzyme, delta-aminolevulinic acid dehydratase (ALAD), and mediate the effect of Pb on this enzyme. Male CD rats were pretreated with 200 mumol Zn/kg, sc, 48 and 24 h prior to assay of renal ALAD, which resulted in activation of renal ALAD and increased the resistance of this enzyme to inhibition by Pb in vitro. To determine the mechanism for this resistance, binding patterns of Zn and Pb in kidney cytosol were assessed. Rats were pretreated with Zn 48 and 24 h prior to injection of 203Pb (170 microCi/kg, ip). Kidneys were removed 4 h later and cytosol was fractionated on a Sephadex G-75 gel filtration column. Both 203Pb and Zn coeluted with the Zn-thionein fraction. Zn-thionein-I and -II, purified previously by DEAE anion-exchange chromatography, bound 203Pb in vitro. In another experiment, addition of purified Zn-thionein to reaction mixtures increased activity of purified bovine liver ALAD twofold and reversed inhibition of ALAD by Pb. Addition of apo-thionein to reaction mixtures partially prevented the inhibition of purified ALAD by Pb, indicating the biological significance of 203Pb chelation. Gel filtration of ALAD assay incubates containing 65Zn-thionein demonstrated that Zn is transferred from Zn-thionein to ALAD. Gel filtration of incubates containing 203Pb demonstrated that the presence of Zn-thionein alters the cytosolic binding pattern of Pb, with less bound to ALAD and more bound to Zn-thionein. The results demonstrate a dual function for Zn-thionein in mediating Pb inhibition of ALAD by a mechanism involving both donation of Zn to this Zn-requiring enzyme and chelation of Pb. These results also suggest that Zn-thionein may serve to regulate ALAD activity in vivo and mediate the inhibition of this enzyme by Pb.
Assuntos
Rim/fisiologia , Chumbo/farmacologia , Metalotioneína/fisiologia , Sintase do Porfobilinogênio/antagonistas & inibidores , Zinco/farmacologia , Animais , Apoproteínas/farmacologia , Bovinos , Chumbo/antagonistas & inibidores , Chumbo/metabolismo , Fígado/enzimologia , Masculino , Ratos , Zinco/metabolismoRESUMO
The bioavailability of lead in kidney is mediated in part by binding to endogenous high-affinity cytosolic lead-binding proteins (PbBP), which are not detectable in liver. Addition of semipurified 11,500 dalton PbBP to liver delta-aminolevulinic acid dehydratase (ALAD) reaction mixtures reverses inhibition of this enzyme by lead and thus provides an explanation for the relative insensitivity of renal ALAD to lead inhibition in vivo and in vitro. This effect results in part from a marked increase in binding of 203Pb to the PbBP relative to control liver cytosol (no PbBP) as demonstrated by Sephadex G-150 gel filtration chromatography. Zinc is known to activate ALAD and is an endogenous component of the PbBP fraction (6 microM in reaction mixtures). Zinc activated hepatic and renal ALAD over a range of 1.5 to 50 microM and also reversed the IC50 lead-inhibited activity. Studies of zinc release and/or displacement from PbBP under ALAD assay conditions (37 degrees C, + glutathione, pH 6.8) were conducted utilizing Sephadex G-25 chromatography. Fifteen to twenty-five percent of the zinc in the PbBP fraction was released, and this value was not markedly influenced by addition of IC50 lead, temperature (4 degrees C) or absence of glutathione; however, zinc release was primarily dependent upon the pH of the reaction mixture. These data indicate that the PbBP fraction attenuates lead inhibition of ALAD in vitro both by chelating lead and apparently serving as a zinc donor for this enzyme under optimal conditions of the ALAD assay.
Assuntos
Proteínas de Transporte/farmacologia , Rim/análise , Chumbo/toxicidade , Sintase do Porfobilinogênio/antagonistas & inibidores , Animais , Fígado/enzimologia , Masculino , Metalotioneína/fisiologia , Peso Molecular , Ratos , Zinco/farmacologiaRESUMO
Selective inhibition of enzymes in the heme biosynthesis pathway with concomitant urinary excretion of heme precursors serve as potentially important biological markers of chemical exposure and cell injury. Intratracheal administration of gallium arsenide particulate suspensions has been shown to result in inhibition of delta-aminolevulinic acid dehydratase (ALAD) in several tissues and increased excretion of the heme precursor aminolevulinic acid (ALA). This study was undertaken to evaluate in vivo the role of gallium alone in ALAD inhibition and increased urinary excretion of ALA. Male CD rats received a single ip injection of Ga2(SO4)3 at doses of 12.5, 25, 50, 100, and 200 mg Ga/kg. A dose-dependent inhibition of ALAD was observed 24 hr later in liver, kidney, and erythrocytes. After injection of 25 mg Ga/kg, maximal inhibition (42 to 49% of control) of ALAD occurred between 6 and 24 hr in liver and kidney with full recovery of activity at 96 hr. In erythrocytes, maximal inhibition (48% of control) occurred between 24 and 48 hr with recovery of activity at 96 hr. Mild to moderate renal proximal tubular necrosis in the pars recta was observed 24 hr after administration of 100 and 200 mg/kg, but no histopathologic changes were evident at lower doses. No consistent changes in urinary excretion of ALA were observed. Lineweaver-Burk analyses of renal and hepatic ALAD activities in the absence and presence of gallium indicated that the inhibition of ALAD by this element is noncompetitive (same Km, decreased Vmax). Gallium was shown to possess an inhibition constant (Ki) of approximately 3 microns for ALAD, similar to the Ki obtained for lead in other studies. Incubation of ALAD in vitro with gallium and lead, an active thiol group inhibitor, resulted in a greater inhibition of the enzyme. Further in vitro studies demonstrated the attenuation of gallium inhibition of hepatic and renal ALAD by zinc, suggesting that the mechanism of gallium action may involve competition for or displacement of zinc from the sulfhydryl group of the enzyme active site. Since ALAD inhibition occurred at doses at which no histopathologic changes were evident, the determination of ALAD activity in various tissues, including blood, may be of potential value as a biomarker of exposure/toxicity to metals such as gallium. The effect of chemical form and route of exposure of gallium and effects of other Group III metals on inhibition of ALAD and excretion of ALA is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Eritrócitos/enzimologia , Gálio/farmacologia , Rim/enzimologia , Fígado/enzimologia , Sintase do Porfobilinogênio/antagonistas & inibidores , Animais , Interações Medicamentosas , Eritrócitos/efeitos dos fármacos , Rim/efeitos dos fármacos , Cinética , Chumbo/farmacologia , Fígado/efeitos dos fármacos , Masculino , Sintase do Porfobilinogênio/metabolismo , Ratos , Ratos Endogâmicos , Zinco/farmacologiaRESUMO
This study was undertaken to evaluate the effect of Zn and Cd pretreatment on the inhibition of delta-aminolaevulinic acid dehydratase (ALAD; porphobilinogen synthase, EC 4.2.1.24) by Pb. Male CD rats were pretreated with 200 mumol of Zn/kg s.c. (subcutaneously) or 18 mumol of Cd/kg s.c., 48 and 24 h before assay of ALAD. Pretreatment with Zn resulted in activation of hepatic and renal ALAD and attenuated the inhibition of this enzyme by Pb in vitro. Pretreatment with Cd increased hepatic ALAD activity, and the inhibitory effect of Pb on the hepatic enzyme was attenuated in this group. In contrast with the situation in liver, pretreatment with Cd did not affect the activity of renal ALAD and did not alter the inhibitory effect of Pb on the renal enzyme. The Pb IC50 (concentration causing half-maximal inhibition) values for hepatic and renal ALAD in Zn-pretreated rats and for hepatic ALAD in Cd-pretreated rats were increased above control, whereas the IC50 for renal ALAD in Cd-pretreated rats was unchanged. Cytosolic binding patterns for the three metals were assessed by gel-filtration chromatography and disclosed that 203Pb was co-eluted with Zn and Cd bound to liver and kidney Zn-thioneins and liver Cd,Zn-thionein, although minimal binding of 203Pb to kidney Cd,Zn-thionein was observed. Estimation of the molar ratio of metals bound revealed Cd/Zn ratios of 2 and 5 for Cd,Zn-thioneins from liver and kidney respectively. The inhibition of purified ALAD by Pb was also attenuated by addition of purified Zn-thioneins and Cd,Zn-thioneins from liver and kidney in the following order: liver Zn-thionein = kidney Zn-thionein greater than liver Cd,Zn-thionein much greater than kidney Cd,Zn-thionein. Thus liver and kidney Zn-thioneins and liver Cd,Zn-thionein with a low Cd/Zn ratio readily decrease the free pool of Pb available to interact with ALAD. These data also demonstrate that the capacity of metallothionein to alter the intracellular distribution of Pb and mediate the inhibition of ALAD by Pb is dependent on the tissue source and relative metal constitution of the metallothionein.
Assuntos
Cádmio/farmacologia , Chumbo/farmacologia , Metalotioneína/farmacologia , Sintase do Porfobilinogênio/antagonistas & inibidores , Zinco/farmacologia , Animais , Cromatografia em Gel , Rim/efeitos dos fármacos , Rim/enzimologia , Fígado/efeitos dos fármacos , Fígado/enzimologia , Masculino , RatosRESUMO
Metallothionein has been postulated to function in essential metal homeostasis. In this study, we demonstrate a 1.7-fold increase in purified bovine liver delta-aminolevulinic acid dehydratase (ALAD) activity following incubation with purified kidney Zn-thionein isolated from Zn-treated rats. The mechanism of enzyme activation, as demonstrated using 65Zn-labeled thionein, involves direct transfer of Zn from Zn-thionein to ALAD. These data support the hypothesis that metallothionein serves to regulate the intracellular bioavailability of essential cations, functioning as a reservoir or conduit through which metals are donated to enzymes which require them as cofactors.
Assuntos
Rim/metabolismo , Metalotioneína/metabolismo , Sintase do Porfobilinogênio/metabolismo , Zinco/metabolismo , Animais , Bovinos , Cromatografia em Gel , Ativação Enzimática/efeitos dos fármacos , Fígado/enzimologia , Ratos , Zinco/farmacologiaRESUMO
The 1976 amendments to the Food, Drug, and Cosmetic Act established requirements for obtaining approval to market new medical devices, including the necessity for demonstrating safety and effectiveness. Evaluation of the potential toxicity of medical devices presents toxicologists with unique problems related to route of exposure, estimation of exposure dose, exposure to by-products generated during manufacture, and matrix effects. A symposium was organized to address the need for toxicologists to examine the potential toxicities of medical device materials, to appreciate the unique problems associated with medical device safety evaluation, to present novel testing methodologies, and to discuss future research needs.
Assuntos
Materiais Biocompatíveis/toxicidade , Equipamentos e Provisões , Animais , Legislação Médica , Fatores de Risco , Estados UnidosRESUMO
Animals pretreated with cadmium (Cd) subsequently develop tolerance to an otherwise lethal dose of Cd. Possible mechanisms for this tolerance include reduced absorption, an altered tissue distribution, and an altered subcellular distribution of Cd. Male rats received a single Cd pretreatment (2.0 mg/kg, sc) 24 hr prior to administration of a typically lethal challenge dose of Cd (3.9 mg/kg, iv). Tolerance was evident because no mortality was observed in Cd-pretreated rats. Since Cd induces synthesis of hepatic metallothionein (MT), a higher percentage of the challenge dose might be sequestered in the liver of Cd-pretreated animals with less distributed to target organs of toxicity. At 2 and 24 hr following Cd challenge, no marked changes in organ distribution of the challenge dose of Cd were observed as a result of Cd pretreatment. However, isolation of hepatic subcellular fractions 2 hr following injection of the challenge dose revealed less Cd in nuclei, mitochondria, and endoplasmic reticulum, and more in cytosol as a result of Cd pretreatment. The increased cytosolic Cd was bound primarily to MT which had been induced markedly by Cd pretreatment. These data indicate that differences in absorption or tissue distribution of Cd are unlikely explanations for development of tolerance to Cd. Rather, tolerance appears to result from an MT-related change in the hepatic subcellular distribution of Cd, evidenced by lower concentrations of Cd in nuclei, mitochondria, endoplasmic reticulum, and cytosolic high-molecular-weight proteins and higher concentrations bound to MT in cytosol.
Assuntos
Cádmio/metabolismo , Animais , Cádmio/toxicidade , Tolerância a Medicamentos , Fígado/metabolismo , Fígado/ultraestrutura , Masculino , Metalotioneína/metabolismo , Proteínas/metabolismo , Ratos , Ratos EndogâmicosRESUMO
The bioavailability of Pb in kidney is mediated in part by binding to high affinity cytosolic Pb-binding proteins (PbBP) of 11,500 (11.5K) and 63,000 (63K) daltons, which are not found in liver. Renal delta-aminolevulinic acid dehydratase (ALAD) is also markedly more resistant to Pb inhibition than hepatic ALAD in vivo. This study was undertaken to evaluate further the differences in sensitivity of renal and hepatic ALAD to Pb and to determine if inhibition of hepatic ALAD by Pb could be reversed by addition of partially purified PbBP from kidney to liver cytosol. Rat liver or kidney cytosol was incubated with Pb over a concentration range of 0.1 to 10 microM. Renal ALAD was 7.5 times more resistant to Pb inhibition than that in liver. Kidney cytosol and 203Pb were incubated before Sephadex G-75 or G-150 column chromatography to isolate the 11.5K and 63K PbBP, respectively. Inhibition of hepatic ALAD activity by Pb was partially reversed by a single addition of semipurified 11.5K PbBP in the presence of 0.1 to 0.4 microM Pb, but no protective effect was observed at higher concentrations of Pb. This effect was not observed with the 63K PbBP added at an equivalent high affinity binding capacity or bovine serum albumin added at an 8-fold higher total protein concentration. Reversal of Pb-induced inhibition of hepatic ALAD activity was dependent on the concentration of 11.5K PbBP in the reaction mixture. Kinetic analysis of either hepatic or renal ALAD activity at an IC50 concentration of Pb indicated a noncompetitive inhibition pattern.(ABSTRACT TRUNCATED AT 250 WORDS)