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

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

Regional protein alterations in rat kidneys induced by lead exposure.

Lead is a potent neuro- and nephrotoxin in humans and a renal carcinogen in rats. Previous studies have detected lead-induced increases in the activities of specific detoxification enzymes in distinct kidney cell types preceding irreversible renal damage. While preferential susceptibility of the highly vascularized cortex to the effects of lead is clear, lead effects on the medullary region have remained unexplored. The present study was undertaken to investigate the extent to which regional renal protein expression differs and to determine which, if any, regionally distinct protein markers indicative of lead's renotoxic mechanism might be detected in kidney cortical and medullary cytosols. We examined protein expression in these two functionally and anatomically distinct regions, and identified several proteins that are differentially expressed in those regions and were significantly altered by lead. Kidney cytosols from rats injected with lead acetate (114 mg/kg, three consecutive daily injections) were separated by two-dimensional electrophoresis. Lead exposure significantly (P<0.001) altered the abundance (either or) of 76 proteins in the cortex and only 13 in the medulla. Eleven of the proteins altered in the protein patterns were conclusively identified either by matrix-assisted laser desorption/ionization mass spectrometry/electrospray ionization-mass spectrometry (MALDI-MS/ESI-MS) analysis of peptide digests, immunological methods, or by gel matching. Several of the cortical proteins altered by lead were unchanged in the medulla while others underwent similar but lesser alterations. These observations reflect the complexity of lead's nephrotoxicity and endorse the application of proteomics in mechanistic studies as well as biomarker development in a variety of toxicologic paradigms.

Differential expression of cytosolic proteins in the rat kidney cortex and medulla: preliminary proteomics.

The rodent kidney is a target of many xenobiotics and is typified by regionally specific structure and function. This renders distinct regions of the kidney differentially susceptible to toxic exposure and effect. To characterize these differences at the proteome level, protein patterns from male rat kidney cortex and medulla cytosols were examined by two-dimensional electrophoresis (2-DE) and image analysis and prominent proteins identified immunologically or by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray/ionization-tandem mass spectrometry (ESI-MS/MS) sequence tag identification. An average of 727 protein spots were resolved and matched to the cortex cytosol reference pattern, and 716 in the medulla. Of this total, 127 proteins were found to differ in abundance (86 higher in cortex; 41 higher in medulla) (P < 0.001). Of those proteins that were detectable in both cortex and medulla, the abundance of 97 differed significantly while 30 proteins were found to be unique to one region or the other (26 in cortex, 4 in medulla). Twenty protein spots were identified and their regional differences are discussed. These results both confirm and expand our understanding of the molecular heterogeneity characterizing structurally and functionally distinct regions of the kidney and serve as a useful foundation for future nephrotoxicologic studies.

Glutathione S-transferases: two-dimensional electrophoretic protein markers of lead exposure.

Glutathione S-transferases (GST) are a family of detoxification isoenzymes that catalyze the conjugation of xenobiotics and their metabolites with reduced glutathione. Lead exposure in rats is known to induce GST isoenzymes in the liver and kidney. These changes in expression have potential use as biomarkers of lead exposure. Because two-dimensional electrophoresis (2-DE) enables one to analyze both protein abundance changes and chemical changes in protein structure, 2-DE was used to determine the effect of in vivo lead exposure on GST isoform expression in rat kidney cytosols. Male Sprague-Dawley rats were exposed to inorganic lead, and proteins were separated by conventional ISO-DALT and NEPHGE-DALT techniques and blotted for immunological identification. Lead exposure caused detectable inductions in both GSTP1 and GSTM1 and quantifiable charge modification in GSTP1. These preliminary data confirm the utility of 2-D electrophoretic GST analysis as indicative of lead exposure and toxicity and support its use for further elaboration of lead's effects on renal protein expression.

Two-dimensional electrophoresis of precision-cut testis slices: toxicologic application.

Advances in tissue slice technology and a recent novel application of this technique to reproductive toxicology using bovine testis have demonstrated the remarkable utility of this approach. The objective of the present study was to combine this in vitro toxicity test system with large-scale two-dimensional polyacrylamide gel electrophoresis (2-DE) to detect and study alterations in testicular-slice protein patterns as molecular correlates of 1,3,5-trinitrobenzene (TNB) and 1,3-dinitrobenzene (DNB) toxicity. Previous studies have shown that testicular slices remain viable for > 24 h and, as measured by protein synthesis inhibition, TNB causes dose-related injury. Tissue-slices were prepared from bovine testicles incubated for 2, 4 or 6 h and exposed to either 100 microM, 500 microM or 1 mM DNB or TNB in the incubation medium. Slices were collected, solubilized, and separated by large scale 2-DE. Resulting protein patterns were then examined by image analysis, which revealed coefficients of variation in protein spot abundance comparable to patterns from fresh rodent tissue samples. Furthermore, specific protein alterations indicated dose-related inductions and declines in protein abundance, some progressive over time. The results of this investigation demonstrate the potential toxicologic utility of combining in vitro tissue-slice technology with high-resolution 2-DE protein mapping. The consolidation of these methods offers a novel approach for toxicity screening and testing, reduces experimental cost, and reduces the use of laboratory animals.

Two-dimensional electrophoretic analysis of myocardial proteins from lead-exposed rabbits.

Despite reported adverse effects, the cardiovascular toxicity of lead remains controversial. The purpose of the present study was to determine if low-level subchronic exposure of rabbits to lead would produce detectable, concentration-dependent changes in myocardial proteins. Lead was administered to male Dutch Belted rabbits as a lead acetate solution, adjusted weekly to achieve and maintain the target blood lead levels of 0, 20, 40, and 80 microg/dL for 15 weeks. Lead exposures did not affect heart or body weights. Myocardial concentrations of lead at sacrifice were 58+/-25, 69+/-23, 102+/-62, and 105+/-37 ng/g. Of 808 individual proteins resolved by two-dimensional electrophoresis (2-DE) in ventricular homogenates, 162 had coefficients of variation < 20%. A number of proteins were tentatively identified based on coordinate positions homologous to other established 2-DE patterns. Despite variable expression of some protein spots, none of the protein abundances analyzed were found to be significantly altered (P < 0.001) by the lead exposures studied. Therefore results show no detectable effect of a low-body burden of lead on major myocardial proteins of the rabbit.

Two-dimensional electrophoretic analysis of compartment-specific hepatic protein charge modification induced by thioacetamide exposure in rats.

Thioacetamide (TA) is a well-known hepatotoxicant. It has been reported that an obligate intermediate of TA binds to proteins with the formation of acetylimidolysine derivatives that are responsible for TA-induced hepatotoxic effects. TA has also been reported to cause chemically induced cell death via both apoptosis and necrosis. The objective of this study was 2-fold: first, to investigate the effect of TA exposure on protein charge modifications in the rat liver and second, to study the role of these molecular correlates in the regulation of cell death. Male Sprague-Dawley rats (200-225 g, 7-8 weeks old) were divided into four major groups and treated intraperitoneally with a 12-fold dose range of TA (50, 150, 300, and 600 mg TA/kg) dissolved in water. Using whole liver extracts, alterations in the hepatic protein pattern following treatment with the 12-fold dose range of TA were studied using high-resolution, two-dimensional polyacrylamide gel electrophoresis and computerized image analysis. The results indicate that charge modification was clearly evident as early as 2 hr with the lowest dose of 50 mg TA/kg. At this dose and time endoplasmic reticulum proteins, calreticulin, grp78, and ER6O exhibited acidic charge variants. The effect of TA became more prominent with dose and time. Generally the elevation of charge modification indices (CMI) by TA appeared to reach a peak between 4 and 6 hr and then while CMI either leveled off or declined in the lower two doses of 50 and 150 mg TA/kg, it continued to remain elevated with the higher doses of 300 and 600 mg TA/kg. This dichotomy in the elevation of CMI is in close correspondence to the pattern of cell death observed with a similar dose range of TA, where lower doses (50 and 150 mg TA/kg) predominantly cause cell death via apoptosis while higher doses cause cell death via necrosis. Delayed charge modification was observed with the cytosolic hsc70s with the 300 and 600 mg TA/kg treatments, indicating that the reactive metabolite(s) slowly leak out into the cytosol from the endoplasmic reticulum. There were no alterations in the mitochondrial proteins hsp60 and grp75, suggesting that TA has no effect on the mitochondrion, its effects primarily being confined to the endoplasmic reticulum. The concept of looking at these proteins as biomarkers of tissue injury has validity. These changes may be indicators of bioactivation and adduct formation and also may be signaling events in the regulation of the mode of cell death.

Influence of growth factors on proliferation and morphogenesis of rabbit ovarian mesothelial cells in vitro.

The ovarian mesothelium (OM) is the source of one of the most frequent and lethal types of common ovarian epithelial tumors, the so-called papillary serous carcinomas. Recent work from our laboratory indicates the existence of postovulatory luteal OM mitogens with variable affinity for heparin. To further investigate the paracrine regulation of this ovarian tissue, rabbit ovarian mesothelial cells (OMC) were cultured in serum-free, fibronectin-rich HL-1 medium with or without one of the following luteal growth factors (0.1, 1, and 10 ng/ml): basic (bFGF) and acidic (aFGF) fibroblastic growth factors, epidermal growth factor (EGF), transforming growth factors alpha (TGF alpha) and beta (TGF beta), tumor necrosis factor alpha, platelet-derived growth factor (PDGF-BB), and vascular endothelial growth factor. After 8 days of culture, OMC growth was stimulated 3-fold by all tested doses of bFGF, EGF, and TGF alpha and 2-2.5-fold by 10 ng/ml of PDGF-BB and aFGF; it was inhibited more than 60% by TGF beta (10 ng/ml). In addition to enhancing the formation of cohesive OMC monolayers, most factors enhanced 3- to 6-fold the aggregation of OMC into papillary processes. The finding of a growth and morphogenetic response to intraluteal growth factors is novel and suggests a role for postovulatory paracrine regulation of OM pathobiology.

Effect of structurally diverse peroxisome proliferators on rat hepatic sulfotransferase.

Exposure to perfluorocarboxylic acids, pthalate esters, and some hypolipidemic agents results in the proliferation of peroxisomes in the rodent liver. The structural diversity of these compounds suggests mechanistic diversity in their toxicity as well. To establish reliable biomarkers of peroxisome proliferation (PP) in compounds with distinct chemical toxicities, this study investigated the effect of in vivo exposure to perfluoro-n-octanoic acid, perfluoro-n-decanoic acid, di(2-ethylhexyl)phthalate (DEHP) and clofibrate on two-dimensional electrophoretic protein patterns of rat hepatic sulfotransferases, ST1A1, ST1C1 and ST2A1. After exposure to peroxisome proliferative doses, both ST1A1 and ST1C1 abundance in whole liver homogenates was significantly reduced, but only as a result of perfluorocarboxylic and exposure. The well-established PPs, DEHP and clofibrate had no effect on sulfotransferase expression whatsoever. The observed down-regulation of these STs is significant with respect to their normal detoxication activities and its potential correlation to carcinogenesis warrants further study. The present investigation supports previous studies that demonstrate the unique features of perfluorocarboxylic acid toxicity, relative to classic peroxisome proliferators and endorses the continued use of 2D protein-mapping of Sts and other proteins as biomarkers of chemical toxicity.

Toxicant-induced alterations in two-dimensional electrophoretic patterns of hepatic and renal stress proteins.

Recent studies in this laboratory and by others suggest that two-dimensional polyacrylamide gel electrophoresis of proteins (2-DE) possesses significant utility in the detection of chemical toxicity and in providing information regarding toxic mechanism. After having identified a set of specific heat-shock and glucose-regulated proteins whose expression in rodent liver and kidney is highly conserved and constitutive, we compared the effect of in vivo exposure to perfluoro-n-octanoic acid and perfluoro-n-decanoic acid on their expression. The following stress proteins were identified, their x, y coordinate positions mapped, and abundance statistically analyzed and compared: hsp32, hsp60, hsc70, hsp70, hsp90, grp75, grp94, protein disulfide isomerase (PDI), and ER60. We report here that the stress response to perfluorocarboxylic acids is tissue-, toxicant-, and stress protein class-specific and dose-related. Furthermore, because nearly all of the proteins studied were constitutively expressed at detectable levels in both liver and kidney, the 2-DE stress protein pattern may be suitable to future toxicologic screening applications.

Ovarian mesothelial and extramesothelial cells in interactive culture.

The ovarian mesothelium (OM) represents the tissue of origin of ovarian epithelial cancer. To gain insight into the regulation of this tissue, OM organoids and submesothelial ovarian stromal cells (SC) were isolated from New Zealand White rabbits by a stepwise tissue dispersal technique, while granulosa cells (GC) were aspirated from mature follicles (14 +/- 4 groups/animal). OM and SC dispersal were sequentially accomplished by: a) 1-h incubation in collagenase type I (300 U/ml), gentle scraping of the ovarian surface, and 1 g sedimentation of OM organoids (equivalent to 0.93 +/- 0.40 x 10(6) cells/animal) on 5% bovine serum albumin (BSA); b) 2-h incubation in pronase-collagenase (0.5%-300 U/ml) under periodical resuspension and gentle scraping of SC (1.40 +/- 0.25 x 10(6)/animal) from OM-denuded ovaries. After a week-long in vitro expansion, OM cells (OMC) were cultured alone and with SC or GC within monocameral vessels or bicameral transfilter vessels in serumless, fibronectinrich (4 micrograms/ml) HL-1 medium. After 7 d of contact cell-cell interaction, cytokeratin-positive OMC became surrounded by fibroblastoid, vimentin-positive SC or by cytokeratin and vimentin-weakly positive GC. Filter-bound OMC humorally interacting with underlying SC or GC displayed a biphasic, epithelioid and spindle, morphology with universal cytokeratin expression. Bromo-2'-deoxyuridine (BrdU) immunoperoxidase revealed mean cell proliferation indices of 14.88% for OMC cultured alone, 11.21% and 19.39% for OMC cultured with GC or SC in monocameral dishes, and 15.25% or 22.47% for OMC cultured in bicameral vessels over GC or SC, respectively. This model provides an experimental tool for investigating the unexplored role of stromal-mesothelial interaction in OM pathobiology.

Two-dimensional electrophoretic mapping of hepatic and renal stress proteins.

Cellular stress proteins and molecular chaperones are responsive to a variety of stressors and therefore comprise an ideal set of proteins with the potential to be used as biomarkers of chemical toxicity. We have investigated the expression of a group of well established heat shock and glucose-regulated proteins in the rat liver and kidney using large-scale two-dimensional protein electrophoresis and computerized image analysis. Our goal was to determine the level of their expression in unstressed target tissues and map their coordinate positions on conventional format two-dimensional electrophoresis (2-DE) gels. All the proteins studied, except for Hsp25 (heat-shock protein) whose expression fell below the level of analyzable detection, were constitutively expressed in liver and kidney. With the exception of Hsp70, all the stress proteins analyzed were constitutively more abundant in the liver than the kidney. Comparison of the sum total of all stress protein abundances revealed a nearly threefold higher level of expression in the liver than the kidney. Our results suggest that this group of proteins has significant responsibilities in normal, unstressed cells, due to their constitutive abundance. Correspondingly, the 2-DE stress protein pattern established in this study may be very useful in toxicologic screening as well as describing a broad range of molecular effects of xenobiotic exposure.

Comparative 2D-electrophoretic mapping of human and rodent hepatic stress proteins as potential biomarkers.

Toxicologic studies in rodents demonstrate that two-dimensional polyacrylamide gel electrophoresis of proteins (2DE) is very useful in the detection and evaluation of chemical toxicity by providing information regarding cellular status at the molecular level. Identification of a set of specific biomarkers of exposure or effect, with a proclivity for both a particular rodent and human target tissue, is required for development of an electrophoretically based testing system. In this regard, stress proteins, such as the heat shock and glucose-regulated proteins (Hsp and Grp), are appropriate candidates. The present investigation was undertaken to identify these stress proteins on conventional two-dimensional electrophoretic gel patterns of human and rat liver homogenates. The following stress proteins were identified, their x, y coordinate positions mapped, and abundances determined, and these data statistically analyzed and compared: Hsp25, Hsp32, Hsp60, Hsc70, Hsp70, Hsp90, Grp75, Grp78, Grp94, protein disulfide isomerase (PDI), and ER-60. With the exception of Hsp25 and Hsp32, the stress proteins examined were constitutively expressed at detectable levels in both unstressed human and rat liver; in virtually identical patterns. Based on our results, the human hepatic 2DE stress protein pattern seems well-suited to toxicologic screening particularly in in vitro applications and via extrapolations from rodent exposures.

Complex fracture-dislocation of the metacarpophalangeal joint. Case report.

A complex fracture-dislocation involving the fifth metacarpophalangeal (MCP) joint occurred in a 16-year-old boy approaching skeletal maturity. Roentgenographic examination demonstrated a widened MCP joint and a dorsally displaced Salter-Harris Type III fracture of the metacarpal head. Attempted manipulative closed reduction was unsuccessful. To achieve successful reduction and fixation it was necessary to make a dorsal approach, incise the interposed volar plate longitudinally, and lever it to the anatomic volar position. Successful fixation of the fracture would have been impossible through a volar approach. The Salter-Harris Type III fracture is a surgical problem.

Characterization of a keratinocyte-derived T cell growth factor distinct from interleukin 2 and B cell stimulatory factor 1.

Epidermal epithelial cells (keratinocytes) produce and secrete a variety of immunologically active cytokines. We have previously reported that both transformed (PAM 212) and normal murine keratinocytes produce a soluble factor which induces proliferation of the T cell line, HT-2. In the present study we sought to compare keratinocyte-derived T cell growth factor (KTGF) with other T cell growth factors, characterize its physicochemical properties, and substantially purify KTGF from PAM 212 conditioned medium. KTGF from PAM 212 conditioned medium was not inhibited by antibodies which block the effect of interleukin 2 (IL 2) (S4B6) or B cell stimulatory factor 1 (BSF 1) (11B11). KTGF is heat-stable, has an isoelectric point of 4.8, and a relative molecular mass of 16 to 23 kilodaltons under nonreducing conditions. KTGF activity was enhanced at least 41,413-fold by sequential hydroxylapatite bulk preparation, desalting by reversed-phase chromatography, gel filtration high pressure liquid chromatography (HPLC), and reversed-phase HPLC. Keratinocytes produce a T cell growth factor with physicochemical properties distinct from IL 2 and BSF 1. KTGF may play a role in regulating the growth and differentiation of T cells in the epidermis.

Single stage lengthening by intercalary bone graft in patients with congenital hand deformities.

The experience with single-stage lengthening by intercalary bone graft in four patients with congenital hand deformities is presented. In all seven lengthenings the gap was bridged by a toe phalanx bone graft. The elongation of the ray ranged from 5-14 mm. Examination of patients followed from one to five years after the treatment showed a good appearance of the lengthened ray. One bone graft assimilation resulted in a pseudoarthrosis and another had osteopenia that resolved spontaneously. All others healed uneventfully. The quality of the skin sensitivity, vascular status and tendon balance is preserved after the lengthening.

Application of an end-systolic pressure-segment length relationship for measuring regional contractility.

A method for estimating regional contractility is described using the end-systolic relationship between left ventricular pressure and myocardial segment-lengths in rapidly volume-loaded beats. The approach was based on the success of previously developed end-systolic relationships between left ventricular pressure and volume and between variable ejection force and fiber length used to describe global contractility in beating hearts. The regional end-systolic relationship was more complicated than its global counterpart, which was load independent, and appeared curvilinear to rapid volume loading. As an approximation of this relationship, a linear slope was constructed between maximum and minimum (pre-ejection) loaded beats of equal cycle length. Because of its load dependency and in order to compare slope relationships between interventions, slope functions were derived only from similarly loaded beats either within or between interventions. Slopes generated by this technique had a reasonable constancy at control conditions and coronary flows with an average SEM of 9.1% of the slope means. End-systolic slopes also appeared sensitive to changes in contractile state, increasing appropriately following treatments with dobutamine and decreasing after propranolol. Following shifts in the end-systolic slopes were unreliable, however, in describing the regional changes in contractility with ischemia. At milder levels of flow restriction, the slopes declined as expected. At moderate levels of flow restriction, the pressure-segment loops shifted markedly rightward and the slope increased. At advanced levels of ischemia, the loops were so distorted, that end-systole could not be identified accurately and the loops essentially described the diastolic compliance characteristics of the left ventricle. Thus the slope estimates of regional contractility as described in this report provided a reliable assessment of inotropic background during modifications with positive and negative inotropic drugs but became invalid as systolic shortening was replaced by aneurysmal bulging during high-grade ischemia.