Mechanical strain at alveolar bone and circummaxillary sutures during acute rapid palatal expansion.

INTRODUCTION: Palatal expansion can potentially affect alveolar bone and circummaxillary sutures. In this study, we characterized their mechanical strain during acute expansion. METHODS: Eight 3- and 6-month-old fresh pig heads received acute palatal expansion with hyrax expanders. Strain gauges were used to measure strain at the buccal alveolar bone of anchor and adjacent nonanchor teeth, and at maxillary-premaxillary, maxillary-zygomatic, and zygomatic-temporal sutures during expansion. Intermolar width changes were measured from dental casts. RESULTS: Intermolar width increased less than expander activation, and the midpalatal sutures were only opened slightly. Alveolar bone strain increased linearly with expander activation and decayed by 20% to 30% during postactivation intervals. Compressive strain at anchor-tooth alveolar bone locations was directed occlusally and apically, related to tooth tipping, and significantly higher than that at nonanchor tooth locations. With expander activation, suture strains increased monotonically and tended to plateau. Suture strain magnitude was generally similar to physiologic (masticatory) strains reported in the literature. The dominant strain polarity was compression at the maxillary-zygomatic and zygomatic-temporal sutures, but there was tension at the maxillary-premaxillary suture. CONCLUSIONS: In these pigs, palatal expansion can cause significant occlusal-apical compression at buccal alveolar bone and physiologic-level strains at circummaxillary sutures.

Effects of short-term zoledronic acid treatment on bone remodeling and healing at surgical sites in the maxilla and mandible of aged dogs.

PURPOSE: It is unknown whether zoledronic acid (ZA) interferes with initial bone healing at extraction and implant sites. The goal of this study was to examine the effect of short-duration ZA on bone remodeling and healing after surgical insult in an aged dog model. MATERIALS AND METHODS: Four 2- to 3-year-old male dogs were administered ZA (0.1 mg/kg per month for 4 months), and 3 age-matched untreated dogs received no drug. In both groups, after the ZA-treated group had completed receiving the drug, the third premolar was extracted unilaterally and 2 orthodontic mini-implants per jaw per dog were placed on the ipsilateral side. After a 6-week healing period, a pair of calcein bone labels were administered. Bone sections from the mandible, maxilla, rib, and femur were obtained. The percent necrosis in the alveolar and basal regions of tooth-supporting bone was assayed by lactate dehydrogenase, and dynamic histomorphometric parameters were quantified and analyzed by use of mixed models. RESULTS: All extraction sites healed uneventfully, and no lesions resembling osteonecrosis were detected. The total percent necrosis was limited to less than 1% for all the bone sites examined. The ZA reduced bone remodeling at both surgical sites (extraction sites and mini-implant site) and nonsurgical sites. Although there was a significant (P < .05) increase in bone formation rate at the surgical sites in the untreated group, this increase was not significant (P = .3) in the ZA-treated group. CONCLUSIONS: Bone remodeling occurs in ZA-treated animals at surgical sites. ZA dramatically reduced bone turnover, but no exposed lesions resembling osteonecrosis developed at extraction and mini-implant sites after the 4-month drug duration.

Relationships of viscosity with contact hardness and modulus of bone matrix measured by nanoindentation.

Creep is an active form of time-dependent viscoelastic deformation that occurs in bone tissue during daily life. Recent findings indicate bone mineralization, which is involved in determining the elastic and plastic properties of bone matrix, can also contribute in controlling its viscoelastic property. Nanoindentation viscosity was used as a direct measure for the capacity of a material to resist viscous-like flow under loading. The objectives of this study were to examine (1) whether the nanoindentation viscosity obtained using the traditional viscoelastic Voigt model can describe creep response of bone matrix and (2) how the nanoindentation viscosity is related to contact hardness and elastic modulus. The Voigt model accurately described the creep behavior of bone matrix (r(2)>0.96, p<0.001). The nanoindentation viscosity had strong relationships with nanoindentation contact hardness (r(2)=0.94, p<0.001) and modulus (r(2)=0.83, p<0.001) independent of tissue ages of osteonal bone matrix. The strong positive relationships of nanoindentation viscosity with contact hardness and modulus can be interpreted as increases in the mineral portion of bone matrix may limit the interfibril motion of collagen while enhancing the mechanical stability of bone. We suggest that previous nanoindentation results can be reanalyzed to characterize the viscoelastic creep using the Voigt model.

Interactive toxicity of inorganic mercury and trichloroethylene in rat and human proximal tubules: effects on apoptosis, necrosis, and glutathione status.

Simultaneous or prior exposure to one chemical may alter the concurrent or subsequent response to another chemical, often in unexpected ways. This is particularly true when the two chemicals share common mechanisms of action. The present study uses the paradigm of prior exposure to study the interactive toxicity between inorganic mercury (Hg(2+)) and trichloroethylene (TRI) or its metabolite S-(1,2-dichlorovinyl)-l-cysteine (DCVC) in rat and human proximal tubule. Pretreatment of rats with a subtoxic dose of Hg(2+) increased expression of glutathione S-transferase-alpha1 (GSTalpha1) but decreased expression of GSTalpha2, increased activities of several GSH-dependent enzymes, and increased GSH conjugation of TRI. Primary cultures of rat proximal tubular (rPT) cells exhibited both necrosis and apoptosis after incubation with Hg(2+). Pretreatment of human proximal tubular (hPT) cells with Hg(2+) caused little or no changes in GST expression or activities of GSH-dependent enzymes, decreased apoptosis induced by TRI or DCVC, but increased necrosis induced by DCVC. In contrast, pretreatment of hPT cells with TRI or DCVC protected from Hg(2+) by decreasing necrosis and increasing apoptosis. Thus, whereas pretreatment of hPT cells with Hg(2+) exacerbated cellular injury due to TRI or DCVC by shifting the response from apoptosis to necrosis, pretreatment of hPT cells with either TRI or DCVC protected from Hg(2+)-induced cytotoxicity by shifting the response from necrosis to apoptosis. These results demonstrate that by altering processes related to GSH status, susceptibilities of rPT and hPT cells to acute injury from Hg(2+), TRI, or DCVC are markedly altered by prior exposures.

Modulation of hepatic and renal metabolism and toxicity of trichloroethylene and perchloroethylene by alterations in status of cytochrome P450 and glutathione.

The relative importance of metabolism of trichloroethylene (Tri) and perchloroethylene (Perc) by the cytochrome P450 (P450) and glutathione (GSH) conjugation pathways in their acute renal and hepatic toxicity was studied in isolated cells and microsomes from rat kidney and liver after various treatments to modulate P450 activity/expression or GSH status. Inhibitors of P450 stimulated GSH conjugation of Tri and, to a lesser extent, Perc, in both kidney cells and hepatocytes. Perc was a more potent, acute cytotoxic agent in isolated kidney cells than Tri but Perc-induced toxicity was less responsive than Tri-induced toxicity to modulation of P450 status. These observations are consistent with P450-dependent bioactivation being more important for Tri than for Perc. Incubation of isolated rat hepatocytes with Tri produced no acute cytotoxicity in isolated hepatocytes while Perc produced comparable cytotoxicity as in kidney cells. Modulation of P450 status in hepatocytes produced larger changes in Tri- and Perc-induced cytotoxicity than in kidney cells, with non-selective P450 inhibitors increasing toxicity. Induction of CYP2E1 with pyridine also markedly increased sensitivity of hepatocytes to Tri but had little effect on Perc-induced cytotoxicity. Increases in cellular GSH concentrations increased Tri- and Perc-induced cytotoxicity in kidney cells but not in hepatocytes, consistent with the role of GSH conjugation in Tri- and Perc-induced nephrotoxicity. In contrast, depletion of cellular GSH concentrations moderately decreased Tri- and Perc-induced cytotoxicity in kidney cells but increased cytotoxicity in hepatocytes, again pointing to the importance of different bioactivation pathways and modes of action in kidney and liver.

Role of organic anion and amino acid carriers in transport of inorganic mercury in rat renal basolateral membrane vesicles: influence of compensatory renal growth.

Susceptibility to renal injury induced by inorganic mercury (Hg(2+)) increases significantly as a result of compensatory renal growth (following reductions of renal mass). We hypothesize that this phenomenon is related in part to increased basolateral uptake of Hg(2+) by proximal tubular cells. To determine the mechanistic roles of various transporters, we studied uptake of Hg(2+), in the form of biologically relevant Hg(2+)-thiol conjugates, using basolateral membrane (BLM) vesicles isolated from the kidney(s) of control and uninephrectomized (NPX) rats. Binding of Hg(2+) to membranes, accounted for 52-86% of total Hg(2+) associated with membrane vesicles exposed to HgCl(2), decreased with increasing concentrations of HgCl(2), and decreased slightly in the presence of sodium ions. Conjugation of Hg(2+) with thiols (glutathione, L-cysteine (Cys), N-acetyl-L-cysteine) reduced binding by more than 50%. Under all conditions, BLM vesicles from NPX rats exhibited a markedly lower proportion of binding. Of the Hg(2+)-thiol conjugates studied, transport of Hg-(Cys)(2) was fastest. Selective inhibition of BLM carriers implicated the involvement of organic anion transporter(s) (Oat1 and/or Oat3; Slc22a6 and Slc22a8), amino acid transporter system ASC (Slc7a10), the dibasic amino acid transporter (Slc3a1), and the sodium-dicarboxylate carrier (SDCT2 or NADC3; Slc13a3). Uptake of each mercuric conjugate, when factored by membrane protein content, was higher in BLM vesicles from uninephrectomized (NPX) rats, with specific increases in transport by the carriers noted above. These results support the hypothesis that compensatory renal growth is associated with increased uptake of Hg(2+) in proximal tubular cells and we have identified specific transporters involved in the process.

Molecular markers of trichloroethylene-induced toxicity in human kidney cells.

Difficulties in evaluation of trichloroethylene (TRI)-induced toxicity in humans and extrapolation of data from laboratory animals to humans are due to the existence of multiple target organs, multiple metabolic pathways, sex-, species-, and strain-dependent differences in both metabolism and susceptibility to toxicity, and the lack or minimal amount of human data for many target organs. The use of human tissue for mechanistic studies is thus distinctly advantageous. The kidneys are one target organ for TRI and metabolism by the glutathione (GSH) conjugation pathway is responsible for nephrotoxicity. The GSH conjugate is processed further to produce the cysteine conjugate, S-(1,2-dichlorovinyl)-l-cysteine (DCVC), which is the penultimate nephrotoxic species. Confluent, primary cultures of human proximal tubular (hPT) cells were used as the model system. Although cells in log-phase growth, which are undergoing more rapid DNA synthesis, would give lower LD(50) values, confluent cells more closely mimic the in vivo proximal tubule. DCVC caused cellular necrosis only at relatively high doses (>100 muM) and long incubation times (>24 h). In contrast, both apoptosis and enhanced cellular proliferation occurred at relatively low doses (10-100 muM) and early incubation times (2-8 h). These responses were associated with prominent changes in expression of several proteins that regulate apoptosis (Bcl-2, Bax, Apaf-1, Caspase-9 cleavage, PARP cleavage) and cellular growth, differentiation and stress response (p53, Hsp27, NF-kappaB). Effects on p53 and Hsp27 implicate function of protein kinase C, the mitogen activated protein kinase pathway, and the cytoskeleton. The precise pattern of expression of these and other proteins can thus serve as molecular markers for TRI exposure and effect in human kidney.

Roles of necrosis, Apoptosis, and mitochondrial dysfunction in S-(1,2-dichlorovinyl)-L-cysteine sulfoxide-induced cytotoxicity in primary cultures of human renal proximal tubular cells.

S-(1,2-Dichlorovinyl)-L-cysteine (DCVC) is the penultimate nephrotoxic metabolite of the environmental contaminant trichloroethylene. Although metabolism of DCVC by the cysteine conjugate beta-lyase is the most studied bioactivation pathway, DCVC may also be metabolized by the flavin-containing monooxygenase (FMO) to yield DCVC sulfoxide (DCVCS). Renal cellular injury induced by DCVCS was investigated in primary cultures of human proximal tubular (hPT) cells by assessment of time- and concentration-dependent effects on cellular morphology, acute cellular necrosis, apoptosis, mitochondrial function, and cellular glutathione (GSH) status. Confluent hPT cells incubated with as little as 10 microM DCVCS for 24 h exhibited morphological changes, although at least 100 microM DCVCS was required to produce marked changes. Acute cellular necrosis did not occur until 48 h with at least 200 microM DCVCS, indicating that this is a high-dose, late response. The extent of necrosis was similar to that with DCVC. In contrast, apoptosis occurred as early as 1 h with as little as 10 microM DCVCS and the extent of apoptosis was much less than that with DCVC. Mitochondrial function was maintained with DCVCS concentrations up to 100 microM, consistent with hPT cells only being competent to undergo apoptosis at early time points and relatively low concentrations. Marked depletion (>50%) of cellular GSH content was only observed with 500 microM DCVCS. These results, combined with previous studies showing protection from DCVC-induced necrosis and apoptosis by the FMO inhibitor methimazole, suggest that formation of DCVCS plays a significant role in trichloroethylene-induced renal cellular injury in hPT cells.

Cellular energetics and glutathione status in NRK-52E cells: toxicological implications.

Cellular energetics and redox status were evaluated in NRK-52E cells, a stable cell line derived from rat proximal tubules. To assess toxicological implications of these properties, susceptibility to apoptosis induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), a well-known mitochondrial and renal cytotoxicant, was studied. Cells exhibited high activities of several glutathione (GSH)-dependent enzymes, including gamma-glutamylcysteine synthetase, GSH peroxidase, glutathione disulfide reductase, and GSH S-transferase, but very low activities of gamma-glutamyltransferase and alkaline phosphatase, consistent with a low content of brush-border microvilli. Uptake and total cellular accumulation of [14C]alpha-methylglucose was significantly higher when cells were exposed at the basolateral as compared to the brush-border membrane. Similarly, uptake of GSH was nearly 2-fold higher across the basolateral than the brush-border membrane. High activities of (Na(+)+K(+))-ATPase and malic dehydrogenase, but low activities of other mitochondrial enzymes, respiration, and transport of GSH and dicarboxylates into mitochondria were observed. Examination of mitochondrial density by confocal microscopy, using a fluorescent marker (MitoTracker Orange), indicated that NRK-52E cells contain a much lower content of mitochondria than rat renal proximal tubules in vivo. Incubation of cells with DCVC caused time- and concentration-dependent ATP depletion that was largely dependent on transport and bioactivation, as observed in the rat, on induction of apoptosis, and on morphological damage. Comparison with primary cultures of rat and human proximal tubular cells suggests that the NRK-52E cells are modestly less sensitive to DCVC. In most respects, however, NRK-52E cells exhibited functions similar to those of the rat renal proximal tubule in vivo.

Renal toxicity of perchloroethylene and S-(1,2,2-trichlorovinyl)glutathione in rats and mice: sex- and species-dependent differences.

Suspensions of renal cells from rats and renal mitochondria from rats and mice were used to assess the sex and species dependence of acute toxicity due to perchloroethylene (Perc) and its glutathione conjugate S-(1,2,2-trichlorovinyl)glutathione (TCVG). A marked sex dependence in the acute cytotoxicity of both Perc and TCVG was observed: Perc caused significant release of lactate dehydrogenase (LDH) in isolated kidney cells from male but not female rats, and TCVG caused much more LDH release from male than female rat kidney cells. Assessment of toxicity in suspensions of isolated mitochondria from kidneys of male and female rats revealed a generally similar pattern of sensitivity, with mitochondria from males exhibiting significantly more inhibition of State 3 respiration and decrease of respiratory control ratio than mitochondria from females. Respiratory function in mitochondria from male and female mice, however, was also significantly inhibited by Perc or TCVG but exhibited little sex dependence in the degree of inhibition. Comparison with results from similar studies using the congener trichloroethylene and its glutathione conjugate suggested that Perc and TCVG are more potent nephrotoxicants. Neither Perc nor TCVG produced any significant effects on cytotoxicity or mitochondrial function in isolated hepatocytes from rats or in isolated liver mitochondria from rats or mice, suggesting that the liver is not a major acute target for Perc or its glutathione conjugate. Thus, many of the species-, sex-, and tissue-dependent differences in toxicity of Perc and TCVG that are observed in vivo are also observed in these in vitro models.